Detection of infectious prion protein by seeded conversion of recombinant prion protein

ABSTRACT

The present disclosure relates to methods and compositions for the detection of infectious proteins or prions in samples, including the diagnosis of prion related diseases. One embodiment is an ultrasensitive method for detecting PrP-res (PrP Sc ) that allows the use of recombinant PrP-sen (rPrP-sen) as a substrate for seeded polymerization. A sample is mixed with purified rPrP-sen to make a reaction mix which is incubated to permit aggregation of the rPrP-sen with the PrP-res that may be present in the sample. Any aggregates are intermittently disaggregated by agitation and the reaction allowed to proceed to amplify target substrate. Any rPrP-res (Sc)  in the reaction mix is detected to indicate the presence of PrP-res in the original sample. In the QUIC method in, the reaction mixture is shaken intermittently. The surprising speed and efficiency of the method permits the rapid identification and diagnosis of prion disease.

PRIORITY

This is a continuation of U.S. patent application Ser. No. 13/489,321,filed on Jun. 5, 2012, which is a continuation of U.S. patentapplication Ser. No. 12/177,012, filed on Jul. 21, 2008, now issued asU.S. Pat. No. 8,216,788, which claims the benefit of U.S. ProvisionalApplication No. 61/021,865, filed Jan. 17, 2008, and U.S. ProvisionalApplication 60/961,364, filed Jul. 20, 2007. All of these priorapplications are incorporated herein by reference in their entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates to methods and compositions for thedetection of infectious proteins or prions in samples, including thediagnosis of prion related diseases.

BACKGROUND

Prion diseases, which are also called transmissible spongiformencephalopathies (TSEs), include a group of fatal infectiousneurodegenerative diseases that include Creutzfeldt-Jakob disease (CJD),kuru, Gerstmann-Strussler Scheinker syndrome (GSS), fatal familialinsomnia (FFI) and sporadic fatal insomnia (sFI) in humans, and scrapie,bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD)in animals. These diseases are characterized by brain vacuolation,astrogliosis, neuronal apoptosis, and the accumulation of misfoldedprion protein (PrP-res, also known as PrP^(Sc) and PrP^(CJD)) in thecentral nervous system. TSEs have incubation periods of months to years,but after the appearance of clinical signs they are rapidly progressive,untreatable, and invariably fatal. Attempts at TSE risk reduction haveled to profound changes in the production and trade of agriculturalgoods, medicines, cosmetics, and biotechnology products.

The hallmark event of prion disease is the formation of an abnormallyfolded protein called PrP^(Sc) (or PrP-res), which is apost-translationally modified version of a normal protein, termedPrP^(C) (also known as PrP-sen). A prion detection method termed proteinmisfolding cyclic amplification (PMCA) is based on the ability of prionsto replicate in vitro in cell lysates containing PrP^(C) (see, forinstance, WO0204954). However, the limitations of PMCA include the timerequired to achieve optimal sensitivity (˜3 weeks) and the requirementfor brain-derived PrP-sen as the amplification substrate.

Castilla et al., Methods in Enzymology 412:3-21 (2006) has stated thatit has not been possible to use PMCA with highly purified prion proteinssuch as PrP^(C). Although the reason for this limitation was unknown, itwas believed that factors in brain homogenates were needed to catalyzeprion propagation. Recombinant PrP-sen expressed from E. coli also lacksglycosylation and the glycophosphatidylinositol (GPI) anchor, which wasadditionally believed to contribute to the difficulty of using rPrP-senin amplification reactions. Such rPrP-sen has been converted toprotease-resistant forms with very limited yields when mixed with PrP inthe past.

Another problem with PMCA is that the formation of PrP^(Sc) reaches aplateau as the number of amplification cycles increases. Castrillon etal. (US Patent Publication No. 2006/0263767) attempted to overcome thisproblem by serial amplification of prion protein by removing a portionof the reaction mix and incubating it with additional non-pathogenicprotein. Although serial amplification PMCA (saPMCA) increases prionamplification and enhances the sensitivity of the assay, the necessityof performing multiple rounds of serial amplification has decreased theoverall practicality of the process.

Supattapone and Deleault (PCT Publication No. WO 2007/082173) also notethat efficiency of amplification may require a cellular factor otherthan PrP-sen. They disclose in vitro amplification of immunoaffinity orexchange chromatography purified PrP-sen in the presence of RNA,synthetic polyanions and partially purified substrates to increase thesensitivity of diagnostic methods for detecting PrP-res.

However, there continues to be a need for a more rapid method for thedetection of PrP-res that is sensitive enough to detect low level prioncontamination. The widespread public health concern about TSE diseasescould be allayed by the development of such a test.

SUMMARY OF THE DISCLOSURE

Disclosed herein is an ultrasensitive method for detecting prion protein(for instance, PrP-res or PrP^(Sc)) that allows the use of recombinantPrP-sen (rPrP-sen) as a substrate for seeded polymerization. Thesemethods include the use of an rPrP-res amplification assay, whichincludes methods such as rPrP-PMCA or QUIC, which differ in the methodused to agitate the reaction. The rPrP-res amplification assays aresurprisingly much faster than existing PMCA methods, yet it stillretains sufficient sensitivity to detect extremely low levels ofPrP-res. The surprising rapidity of the method permits the rapididentification and diagnosis of prion disease, which can limit thetransmission of prion diseases, particularly through the food supply.

One embodiment of the disclosure is a method for detecting PrP-res(PrP^(Sc)) in a sample. The method includes the steps of (a) mixing thesample with purified rPrP-sen to make a reaction mix, and (b) performingan amplification reaction between PrP-res (PrP^(Sc)) and rPrP-sen in themixture that results in the formation and amplification of one or morespecific forms of recombinant PrP-res (for instance, rPrP-res^((Sc)).The amplification reaction includes the steps of (i) incubating thereaction mix to permit co-aggregation or co-polymerization of therPrP-sen with the PrP-res that may be present in the reaction mix, and(ii) agitating any aggregates or multimers formed during step (i), forinstance by shaking or sonication, and (iii) repeating steps (i) and(ii) one or more times. In step (i), aggregation of the rPrP-sen withany PrP-res that may be present in the sample results in a conversion ofthe rPrP-sen to rPrP-res^((Sc)). After the amplification reaction iscarried out, rPrP-res^((Sc)) is detected in the reaction mix as anamplified indicator of any PrP-res originally present in the sample.This amplification procedure can be performed on an initial sample ofinterest, such that the method is preformed only as a single round ofamplification. Optionally, a serial amplification reaction is carriedout with the same steps as the first round reaction, except an aliquotof the amplified reaction mixture (instead of the original sample) ismixed with purified rPrP-sen.

In particular embodiments, the amplification reaction is carried outunder conditions that inhibit production of spontaneously aggregatedrPrP-res (rPrP-res^((spon))) that is independent of the presence ofPrP-res in the sample, because that by-product has surprisingly beenfound to interfere with the desired aggregation reaction of rPrP withPrP-res and can complicate the detection of rPrP-res^((Sc)). Inhibitingthe production of the by-product increases the speed, sensitivity, andreliability of the amplification reaction.

In the embodiment referred to as the QUIC assay, agitation of aggregatesto disaggregate them is carried out in multiple-container trays that arephysically shaken without sonication to transmit the disaggregatingenergy substantially equally to all the containers in the tray. The useof shaking instead of sonication has been found to provide a more easilyduplicated and rapid test that retains a high degree of sensitivity.

The foregoing and other features and advantages of the disclosure willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a series of digital images of gels showing the comparison ofhamster proteinase K resistant prion protein (HaPrP^(Sc))-seeded andunseeded recombinant hamster proteinase K-sensitive prion protein(rHaPrP-sen) conversion reactions. FIG. 1A is a digital image of a gelcomparing the results of the assay when a designated amount of purifiedHaPrP^(Sc) was incubated with 0.2 mg/ml rHaPrP-sen in 0.1% sodiumdodecyl sulphate (SDS) and 0.1% TX-100 in phosphate buffered saline(PBS) for 24 hours, with (lanes 5-8) or without (lanes 1-4) periodicsonication (a 40-second pulse every hour). rHaPrP-sen was omitted fromreactions shown in lanes 1 and 5. The reactions were digested withproteinase K (PK; 0.025:1 PK/rPrP weight/weight) and equivalent aliquotswere subjected to immunoblotting using the polyclonal antibody R20,which was raised against prion protein residues 219-232. Open circlesand black diamonds mark the 17- and 10-kDa fragments, respectively. FIG.1B is a digital image of a gel showing the results of the assay whenaliquots of first round HaPrP^(Sc)-seeded, sonicated reaction productsshown in lane 7 of FIG. 1A were diluted by the designated factors intofresh rHaPrP-sen and subjected to a second round of sonicated orunsonicated reactions and PK treatments as in FIG. 1A. Lanes designated“No seed” indicate reactions that were left unseeded. FIG. 1C is aseries of digital images of three gels showing the antibody reactivityof PK-treated reaction products, which was determined after threesequential rounds of reactions seeded in the first round with 0 or 40 ngPrP^(Sc). The reactions were diluted 100-fold into fresh rHaPrP-senbetween each round. The third round reactions were digested with thedesignated PK:PrP ratios and analyzed by immunoblot with D13, R18 andR20 antibodies. The respective antibody epitopes are contained withinthe prion protein residues indicated in parentheses. Lanes 1 and 5 show2 μl samples (400 ng of rHaPrP) without PK digestion. Lane 9 is 100 ngrHaPrP-sen without PK digestion. Asterisks indicate dimer formed from12-13 kDa fragments, suggested by their size and lack of recognition byD13, an antibody which would react with full-length rPrP but not with adimer of 13-kDa fragments containing the C-terminal epitope of R20. FIG.1D is a digital image showing silver staining of rHaPrP-res^((Sc)) orunseeded (rHaPrP-res^((spon)) third-round after PK digestion (0.025:1PK/rPrP). Positions of molecular mass markers are designated in kDa.

FIG. 2 is a pair of digital images of gels showing the detection limitsof rPrP-protein misfolding cyclic amplification (rPrP-PMCA). FIG. 2A isa pair of digital images showing the results of the first round ofrPrP-PMCA. Serially diluted scrapie brain homogenate (ScBH) containingthe designated amounts of PrP^(Sc) was used as seeds. Normal brainhomogenate (NBH) (1%) was used for negative controls (lanes 8-10) and asa diluent for the ScBH. The reactions seeded with 2-50 ag of PrP^(Sc) orNBH were done in triplicate. Untreated rHaPrP-sen is shown in lanes 1and 11. All other samples were treated with PK (0.025:1 PK/rPrP wt/wtratio) for 1 hour at 37° C. Samples were probed with anti-PrP monoclonalantibody D13. FIG. 2B is a pair of digital images showing the results ofthe second round of rPrP-PMCA. One tenth volume (8 μl) of the firstround samples was transferred to a newly prepared substrate mixture. PKdigestion and immunoblotting were done as described in Example 1.Similar results were obtained in another independent experiment.Positions of molecular mass markers are designated in kDa.

FIG. 3 is a pair of digital images of gels showing seeding competitionbetween rHaPrP-res^((Sc)) and rHaPrP-res^((spon)). Purified HaPrP^(Sc)and rHaPrP-res^((spon)) were each used to initiate three successiverounds of rPrP-PMCA. Aliquots of the third-round reactions containingsimilar amounts of either rHaPrP-res^((Sc)) and rHaPrP-res^((spon)) wereused to seed fourth round reactions, which were subjected to sonicationcycles over 24 hours as described in Example 2. The relative seedamounts of 1, 10 and 100 designate reactions seeded with 0.08, 0.8 or 8μl, respectively, of the final third-round reaction volume. PK-treatedreaction products of the third-round (FIG. 3A) and fourth-round (FIG.3B) reactions were analyzed by immunoblotting with antiserum R20. The17-kDa and 10-kDa bands specific for the rHaPrP-res^((Sc))- andrHaPrP-res^((spon))-seeded reactions, respectively, are marked with anopen circle and a diamond, respectively. Positions of molecular massmarkers are designated in kDa.

FIG. 4 is a pair of digital images of gels showing the results ofseeding rPrP-PMCA with cerebrospinal fluid (CSF). Aliquots (2 μl) of CSFtaken from normal hamsters (n=3) or hamsters in the clinical phase ofscrapie (n=6) were used to seed rPrP-PMCA reactions Immunoblots of thePK-digested products of the first 24-hour round are shown in FIG. 4A.Ten percent of each first round reaction volume was used to seed asecond 24-hour round of rPrP-PMCA and the PK-digested products of thelatter are shown in FIG. 4B. Antisera D13 and R20 were used for theimmunoblots. Lane 1 of each panel shows 100 ng HaPrP-sen without PKtreatment. The rPrP-PMCA reaction products were digested with a PK:PrPratio of 0.025:1 (w/w). The positions of the 17-kDa rHaPrP-res^((Sc))band are marked with a circle.

FIG. 5 is a pair of digital images of gels and a graph showing thegeneration of thioflavin-T positive, protease resistant recombinantmouse prion protein (rMoPrP) fragments by sonication. FIG. 5A is a pairof digital images of gels showing the results of rPrP-PMCA. Solutions ofrMoPrP (0.4 mg/ml, 16 μM) in PBS pH 7.4, and SDS (0-0.5%) were preparedin 100 μL volumes. The tubes were incubated at 37° C. in a cuphornsonicator bath. The samples were then subjected to repeated cycles of 9minutes of incubation followed by 1 minute of sonication at 100% power.After 18 hours, the samples were treated with PK. PK-digested sampleswere immunoblotted with antibody R20. Upper and lower panels correspondto incubations without and with sonication, respectively. Lane 1 of eachpanel shows 100 ng of rHaPrP-sen without PK digestion. Molecular massmarkers are indicated in kilodaltons on the left side. FIG. 5B is agraph showing the kinetics of increase in the fluorescence of theamyloid stain thioflavin T when combined with sonicated or unsonicatedsamples of rMoPrP in 0.1% SDS as in FIG. 5A. Thioflavin T (ThT)fluorescence typically increases upon interaction with amyloid fibrils(Prusiner (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13363-13383).Aliquots (5 μl) were withdrawn at each time point and diluted into 10 μMthioflavin T, 50 mM glycine pH 8.5 to a volume of 100 μl. Fluorescenceemission was measured at 482 nm with excitation at 445 nm. Threeindependent reactions with sonication are shown relative to a singlecontrol reaction done without sonication.

FIG. 6 is a series of digital images of gels and graphs showing theresults of seeding reactions with sonicated rPrP-res under unsonicatedconditions. FIG. 6A is a pair of digital images of immunoblots showingproducts of unsonicated conversion reactions that were either unseededor seeded with 1.6 μl aliquots of sonicated reactions containingrMoPrP-res(spon) ([Mo]) or rHaPrP-res(spon) ([Ha]) and total prionprotein concentrations of 0.4 mg/ml. The seed volumes were added to 80μL 0.4 mg/ml rMoPrP-sen or rHaPrP-sen in 0.1% SDS, 10 μM thioflavin T(ThT) and PBS, pH 7.4, in 96-well assay plates. The reactions wereincubated for 96 hours without sonication. Aliquots were digested withPK at the designated PK:rPrP ratio and analyzed by immunoblotting withantibody R20. The first lane of each panel shows 100 ng of rPrP-senwithout PK treatment. FIG. 6B is a pair of graphs showing the kineticsof reactions seeded with the designated % volumes ofrMoPrP-res(spon)-containing reaction products, followed by monitoringThT fluorescence at 482 nm (left graph; data points are means±SD, n=3).The results of heterologous reactions in which rMoPrP-res(spon) was usedto seed the conversion of rHaPrP-sen are also shown. The right graphshows the linear relationship between seed concentration and ThTfluorescence (r2=0.998) after 32 hours under these unsonicated reactionconditions. FIG. 6C is a pair of graphs showing the kinetics ofanalogous homologous and heterologous reactions seeded withrHaPrP-res(spon)-containing reaction products. The right graph shows thelinear relationship between the amount of seed and ThT fluorescenceafter 8 hours (r2=0.997).

FIG. 7 is a pair of digital images of gels showing the effects of SDSand Sarkosyl upon treatment of rPrP-PMCA reaction products with highconcentrations of PK. Aliquots of third round PrPSc-seeded or unseededrPrP-PMCA reaction products containing either rHaPrP-res^((Sc)) (Sc) orrHaPrP-res(spon) (spon) were treated with 20 μg/ml PK (PK:PrPratio=0.5:1) as described in Example 1 except for the addition of thedesignated concentrations of SDS or Sarkosyl. This PK concentration is20-fold higher than used in most of the other experiments describedherein. This stronger PK treatment in 0.1-2% SDS severely reduced therelative recovery of the characteristic 17 kDa rHaPrP-res^((Sc)) band(compare to FIG. 5B, lanes 2-7, for example). However, 1-2% Sarkosylstrongly enhanced the recovery of the 17-kDa rHaPrP-res^((Sc)) bandwhile retaining striking differences between the banding profiles ofrHaPrP-res^((Sc)) and rHaPrP-res^((spon)). Therefore, the addition ofSarkosyl together with higher concentrations of PK can provide rPrP-PMCAdigestion conditions that are more robust and less sensitive to minorvariations in PK activity or total protein concentrations of thereaction mixtures.

FIG. 8 is a series of digital images of electron micrographs showing theultrastructure of rHaPrP-res^((Sc)) (FIGS. 8A, 8C, 8E) andrHaPrP-res^((spon)) (FIGS. 8B, 8D, 8F). To further characterize thestructure of rHaPrP-res^((Sc)) and rHaPrP-res^((spon)), the samples wereexamined with transmission electron microscopy. Electron micrographs ofboth samples prior to PK digestion revealed thick overlapping fibrebundles, the definition and edges of which were somewhat blurred (FIGS.8A, 8B). After PK digestion (FIGS. 8C, 8D), the fibrils within thesebundles were better resolved, indicating that the PK resistant cores ofthe fibrils are coated with PK sensitive material, either the rHaPrP-senthat has yet to convert to a more resistant structure, the flexibleN-termini projecting outwards, or both. In some instances more separatedfibrils could be detected, although their tendencies to cluster togethergave false impressions of increased width when viewed without furthermagnification. Storing the material in water further dissociated thebundles, yielding more clearly defined fibril clusters for comparison(FIGS. 8E, 8F). Widths of fibrils at their thinnest were approximately2-3 nm. The rHaPrP-res^((spon)) fibrils preferentially clustered in whatappeared to be doublets, with total widths of 6-8 nm, while those ofrHaPrP-res^((Sc)) formed larger side by side clusters of up to 36 nm inwidth. Bars designate 100 nm.

FIG. 9 is a series of graphs showing Fourier transform infraredspectroscopy (FTIR) spectroscopy of rHaPrP-res^((Sc)) andrHaPrP-res^((spon)). To compare the secondary structures ofrHaPrP-res^((Sc)) and rHaPrP-res^((spon)), samples were prepared usingthree sequential rounds of rPrP-PMCA so that the original HaPrP^(Sc)remaining in the seeded sample was <0.0001% of the total prion proteinanalyzed. Portions of each sample were left undigested (FIG. 9A) ordigested with PK (FIG. 9B) and analyzed by FTIR. The spectrum of therHaPrP-sen substrate is shown for comparison. Overlaid spectra are fromindependent preparations. As expected, rHaPrP-sen had an absorbancemaximum at ˜1652 cm⁻¹, consistent with prominent α-helical and/ordisordered secondary structures. In contrast, both rHaPrP-res^((Sc)) andrHaPrP-res^((spon)) displayed prominent bands at lower wavenumbers(1615-1628 cm⁻¹), indicating higher proportions of β-sheet. However, thelocation of the bands differed between the two types of rHaPrP-res.Without PK treatment, the rHaPrP-res^((Sc)) had maxima at 1628 and 1615cm⁻¹, whereas rHaPrP-res^((spon)) peaked at 1625 cm⁻¹. After PKdigestion of both types of rHaPrP-res, the intensities of bands in theregion associated with the α-helix and/or disordered structures wereattenuated. Prominent differences remained between rHaPrP-res^((Sc)),with maxima at 1659, 1647, 1637 and 1628 cm⁻¹, and rHaPrP-res^((spon)),with maxima at 1664 and 1627 cm⁻¹. These spectral differences could bedue to differences in conformation, PK-resistant polypeptide chainlength, or both. Precise assignments of these bands are uncertain, butthe 1664 cm⁻¹ band is often associated with turns, and the 1659 and 1647cm⁻¹ bands with loops or helices, and disordered structures,respectively. Of particular interest is the 1637 cm⁻¹ band ofPK-digested rHaPrP-res^((Sc)). This band also features prominently inthe spectrum of 263K HaPrP^(Sc) (spectrum of PK-treated sample is shownin FIG. 9B) and is absent from the spectrum of the DY strains ofHaPrP^(Sc), indicating that strain-dependent structure associated withthe 1637 cm⁻¹ band in 263K HaPrP^(Sc) was replicated inrHaPrP-res^((Sc)). This provides further evidence of the conformationalfidelity of rPrP-PMCA amplification.

FIG. 10 is a series of digital images of gels showing that tube shakingsupports ultra-sensitive prion-seeded conversions of rPrP-sen. PurifiedPrPSc (FIG. 10A) or scrapie brain homogenate (FIG. 10B) were used toseed the conversion of rHaPrP-sen to protease-resistant forms in QUICreactions performed in 0.1% sodium dodecyl sulfate (SDS) and 0.1% TritonX-100 (C₁₄H₂₂O(C₂H₄₀)_(n), also known as octylphenoxypolyethoxyethanol;Octoxynol-9; 4-octylphenol polyethoxylate; or polyethylene glycolp-(1,1,3,3-tetramethylbutyl)-phenyl ether, octyl phenol ethoxylate,polyoxyethylene octyl phenyl ether), in PBS. PK digestions andimmunoblotting of reaction aliquots were performed as described inExample 8. The C-terminal polyclonal antibody R20 was used in theimmunoblots. Circles designate the 17-kDa rHaPrP-res(Sc) band andbrackets designate the position of the ≦13 kDa rHaPrP-res(Sc) bands.FIG. 10A shows a comparison of PK-resistant QUIC reaction products fromduplicate 24-hour unshaken reactions and reactions shaken with orwithout 0.1 mm glass cell disruption beads (Scientific Industries). 50μl reactions were seeded containing 0.1 mg/ml (4 μM) hamster rPrP-senwith 10 ng of purified hamster PrPSc and subjected the tubes to cyclesof 2 minutes of shaking and 28 minutes without shaking at 37° C.Enhanced rHaPrP-res(Sc) formation was noted in the shaken reactions, butthe beads were not influenced. 100 ng of rPrP-sen without PK-treatmentis shown in lane 1. FIG. 10B shows 20-hour QUIC reactions performed withthe designated rPrP-sen concentrations, reaction volumes, and seedamounts. The seed amounts indicate the estimated quantity of PrPSc addedin 2-μl aliquots of scrapie brain homogenate diluted in 1% normal brainhomogenate. Lanes 6, 12, 18, and 24 received aliquots of only 1% normalbrain homogenate. The tubes were subjected to cycles of 10 seconds ofshaking and 110 seconds without shaking. The asterisk marks the positionof rHaPrP-res(spon) bands.

FIG. 11 is a pair of digital images of gels showing that extendedreactions can enhance QUIC sensitivity to small amounts of scrapie brainhomogenate seed. QUIC reactions were performed with 0.1 mg/ml rPrP-senand the designated reaction volumes and seed amounts using the shakingcycle and buffer conditions described for FIG. 10B. Two digital imagesare shown, 65-hour (upper blot) and 95-hour (lower blot) QUIC reactionswere performed as using 100-μl reaction volumes and dilutions of scrapiebrain homogenate containing the designated amount of PrP^(Sc). The lanesmarked ‘none’ received comparable amounts of normal brain homogenateonly. Antiserum R20 was used for these blots. Open circles designate the17-kDa rHaPrP-res^((Sc)) band and brackets designate the positions ofthe 10-13 kDa rHaPrP-res^((Sc)) or rHaPrP-res^((spon)) bands. Thepositions of molecular mass markers are designated in kDa on the left.

FIG. 12 is a digital image of a gel showing the results of serial QUICreactions. For the first round, QUIC reactions were performed under theconditions described in the brief description of FIG. 11B, except forthe use of 48-hour reaction times and reduced detergent concentrations(0.05% SDS and 0.05% Triton X-100). For the second round, 10% of thevolume of the first round reaction products were diluted into 9 volumesof reaction buffer containing fresh rPrP-sen. PK-digested products wereimmunoblotted using D13 primary antibody. Open circles designate the17-kDa rHaPrP-res^((Sc)) band. The positions of molecular mass markersare designated in kDa on the left.

FIG. 13 is a digital image of gels showing the results of seeding QUICreactions with CSF. Aliquots (2 μl) of CSF taken from normal hamsters(n=3) or hamsters in the clinical phase of scrapie (n=6) were used toseed QUIC reactions using the conditions described for FIG. 12.Immunoblots of the PK-digested products of the first 48-hour round areshown in FIG. 13A. Ten percent of each first round reaction volume wasused to seed a second 48-hour round of QUIC and the PK-digested productsof the latter are shown in FIG. 13B. Antibodies R20 (top) and D13(bottom) were used for the immunoblots. Lane 1 of each panel shows 100ng HaPrP-sen without PK treatment. The positions of the 17-kDarHaPrP-res^((Sc)) band are marked with a circle. The positions ofmolecular mass markers are designated in kDa on the left. These 37° C.reactions contained 0.05% SDS and 0.05% Triton X-100 in PBS and wereshaken at 1500 rpm for 10 seconds every 2 minutes. FIG. 13A showsimmunoblots with antibody R20 of the PK-digested products of the first48-h round. FIG. 13B is an R20 immunoblot showing products ofsecond-round reactions seeded with 10% of each first round reactionvolume.

FIG. 14 is a digital image of gels showing ultrasensitive prion-seededconversions of rPrP-sen in single-round 46-hour QUIC reactions at 45° C.Scrapie brain homogenate was used to seed the conversion of rHaPrP-sento protease-resistant forms in QUIC reactions (0.1% SDS and 0.1% TritonX-100, in PBS). The reactions were shaken at 1500 rpm for 10 s every 2min PK digestions and immunoblotting of reaction aliquots were performedwith the C-terminal antibody R20. Circles designate the 17-kDarHaPrP-res (Sc) band and brackets designate the position of the ≦13 kDarHaPrP-res(Sc) bands. FIG. 14A illustrates the sensitivity of thereaction with dilutions of normal brain homogenate (NBH) and scrapiebrain homogenate (ScBH) as seeds. The ScBH seeds contained thedesignated amounts of PrPSc. The NBH was 0.00001% w/v in the reaction,which is equivalent to that of the ScBH seed dilution containing 1 pg ofPrPSc. The NBH and ScBH samples were diluted to the designated levels in1% N-2 supplement (Invitrogen), except in the lanes marked 1 pg*, whichwere diluted in 0.1% N-2. Either NBH or N-2 can be used as a diluent.FIG. 14B is an analysis of multiple negative controls under thereactions conditions of FIG. 14A. The ScBH seeds contained 1 pg of PrPScwhile the NBH content in the negative controls are as designated. Thelanes marked none were seeded with the diluent for the brainhomogenates, i.e., N-2. Molecular mass markers are designated on theleft.

FIG. 15 is a pair of digital images of gels showing that extendedreactions can enhance QUIC sensitivity to small amounts of scrapie brainhomogenate seed. In FIG. 15A, 40-hour QUIC reactions were performed with0.1 mg/ml rPrP-sen and the designated reaction volumes and seed amountsusing the shaking cycle and buffer conditions described for FIG. 10B.The upper and lower panels show immunoblots performed using antibody R20and D13, respectively (PrP epitope residues shown in parentheses). InFIG. 15B, 65-hour (upper blot) and 95-hour (lower blot) QUIC reactionswere performed as in FIG. 15A using 100-μl reaction volumes anddilutions of scrapie brain homogenate containing the designated amountof PrP^(Sc). The lanes marked ‘none’ received comparable amounts ofnormal brain homogenate only. Antiserum R20 was used for these blots.Open circles designate the 17-kDa rHaPrP-res^((Sc)) band and bracketsdesignate the positions of the 10-13 kDa rHaPrP-res^((Sc)) orrHaPrP-res^((spon)) bands. The positions of molecular mass markers aredesignated in kDa on the left. FIG. 11 and FIG. 15B provide results fromthe same experiment.

FIG. 16 is a series of digital images showing the effect of temperatureon QUIC reaction products and kinetics. QUIC reactions were seeded atdifferent temperatures and reaction times with scrapie brain homogenates(diluted in N2) containing the designated amount of PrP^(Sc) or normalbrain homogenate (NBH) at the dilution used for the 100-fg scrapie brainhomogenate sample. The PK-digested products were immunoblotted withantibody R20. Rows FIG. 16A, 16B, 16C, and 16D show reactions performedat 37° C., 45° C., 55° C. and 65° C., respectively. Successive columnsof blots show reactions run for 4, 8 and 18 hours. All of the QUICreactions were run in 0.1% SDS and 0.1% Triton X-100 in PBS with 0.1mg/ml rPrP-sen with 60 seconds of shaking at 1500 rpm and 60 seconds ofrest. The reaction products were digested with PK under theSarkosyl-containing conditions described in Example 8. The positions ofmolecular mass markers are designated in kDa on the left in the firstcolumn or by corresponding tick marks by the other columns. The opencircles designate the position of the 17 kDa band and the bracket the10-13 kDa bands.

FIG. 17 illustrates the effect of shaking variations on the QUICreaction. QUIC reactions were subjected to cycles of 10 seconds shakingand 110 seconds res (FIG. 17A) with reactions shaken for 60 seconds andrested for 60 seconds (FIG. 17B). These reactions were seeded withscrapie brain homogenate (NBH) at dilutions identical to that used forthe 10 fg scrapie brain homogenate sample. The reaction temperature was45° C. and the QUIC buffer conditions, PK-digestion and immunoblotprotocols were as described for FIG. 18. The positions of molecular massmarkers are designated in kDa on the left; the open circles designatethe position of the 17 kDa band and the bracket the 10-13 kDa bands.

FIG. 18 illustrates the effect of detergent conditions on PK digestionof QUIC reaction products. QUIC reactions performed at 45° C. wereseeded with scrapie brain homogenates (diluted in N2) containing 100 fgof PrP^(Sc) or the same dilution of normal brain homogenate (NBH). Theshaking cycle was 10 seconds on and 110 second off, and the bufferconditions were as described in connection with FIG. 16. 10-μl aliquotsof the reaction products were mixed with 4 μl of the designateddetergent solutions and digested with 7 μg/ml PK (final concentration)for 60 minutes at 37° C. The samples were then immunoblotted using R20antibody. The positions of molecular mass markers are designated in kDaon the left; the open circles designate the position of the 17 kDa bandand the bracket the 10-13 kDa bands. The upper band represents residualfull length rPrP molecules.

FIG. 19 is a digital image of a blot showing the sensitivity of an18-hour QUIC reaction at 55° C. QUIC reactions were seeded with scrapiebrain homogenates (diluted in N2) containing the designated amount ofPrP^(Sc) or normal brain homogenate (NBH) at the dilution used for the10-fg scrapie brain homogenate sample. Reaction buffer constituents,PK-digestion conditions, and immunoblotting were as described in thelegend to FIG. 12. The positions of molecular mass markers aredesignated in kDa on the left. The open circles designate the positionof the 17 kDa band and the bracket the 10-13 kDa bands.

FIG. 20 shows blots from a QUIC reaction seeded either with dilutions ofbrain homogenate from a variant CJD patient containing 100 fg, 10 fg, or1 fg of PrP-res or, as a negative control, a dilution of a non-CJD humanbrain homogenate (from an Alzheimer's disease patient) equivalent to the100-fg vCJD brain homogenate dilution. The recombinant PrP substrate inthese reactions was the Syrian hamster PrP sequence (residues 23-231).This was a single-round reaction at 50° C. for either 8 hours (topblots) or 18 hours (bottom blots). The primary antibody used to detectthe rPrP-res[CJD] reaction products was monoclonal Ab 3F4, which has anepitope within residues 106-112, and thus, is only expected to detectthe 17-kDa rPrP-res[CJD] product and not the smaller bands that aredetected by more C-terminally reactive antibodies. Six separatereactions were performed with each type or dilution of seed and thenumber of rPrP-res[CJD]-positive reactions per 6 replicates is indicatedbelow each set of replicates on the blots.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file4239-77856-08_Sequence_Listing.txt, Apr. 28, 2014, ˜21.8 KB], which isincorporated by reference herein.

The nucleic acid sequences listed in the accompanying sequence listingare shown using standard letter abbreviations for nucleotide bases, asdefined in 37 C.F.R. 1.822. Only one strand of each nucleic acidsequence is shown, but the complementary strand is understood asincluded by any reference to the displayed strand. In the accompanyingsequence listing:

SEQ ID NO: 1 is an amino acid sequence of a recombinant Syrian golden hamster proteinase  K-sensitive prion protein.kkrpkpgg wntggsrypg qgspggnryp pqgggtwgqp hgggwgqphg ggwgqphggg wgqphgggwg qgggthnqwn kpnkpktsmk hmagaaaaga vvgglggyml gsamsrpmlh fgndwedryy renmnrypnq vyyrpvdqyn nqnnfvhdcv nitikqhtvt tttkgenfte tdvkmmervv eqmcvtqyqk  esqayydgrr sSEQ ID NO: 2 is an amino acid sequence of a recombinant mouse (Prnp-a) proteinase   K-sensitive prion protein.kkrpkpgg wntggsrypg qgspggnryp pqggtwgqph gggwgqphgg swgqphggsw gqphgggwgq gggthnqwnk pskpktnlkh vagaaaagav vgglggymlg samsrpmihf gndwedryyr enmyrypnqv yyrpvdqysn qnnfvhdcvn itikqhtvtt ttkgenftet dvkmmervve qmcvtqyqke  sqayydgrrsSEQ ID NO: 3 is an amino acid sequence of a recombinant human (129M) proteinase  K-sensitive prion protein.kkrpkpgg wntggsrypg qgspggnryp pqggggwgqp hgggwgqphg ggwgqphggg wgqphgggwg qgggthsqwn kpskpktnmk hmagaaaaga vvgglggyml gsamsrpiih fgsdyedryy renmhrypnq vyyrpmdeys nqnnfvhdcv nitikqhtvt tttkgenfte tdvkmmervv eqmcitqyer  esqayyqrgs sSEQ ID NO: 4 is an amino acid sequence of a recombinant human (129V) proteinase  K-sensitive prion protein.kkrpkpgg wntggsrypg qgspggnryp pqggggwgqp hgggwgqphg ggwgqphggg wgqphgggwg qgggthsqwn kpskpktnmk hmagaaaaga vvgglggyvl gsamsrpiih fgsdyedryy renmhrypnq vyyrpmdeys nqnnfvhdcv nitikqhtvt tttkgenfte tdvkmmervv eqmcitqyer  esqayyqrgs sSEQ ID NO: 5 is an amino acid sequence of a recombinant bovine (6-octarepeat) proteinase  K-sensitive prion protein.kkrpkp gggwntggsr ypgqgspggn ryppqggggw gqphgggwgq phgggwgqph gggwgqphgg gwgqphgggg wgqggthgqw nkpskpktnm khvagaaaag avvgglggym lgsamsrpli hfgsdyedry yrenmhrypn qvyyrpvdqy snqnnfvhdc vnitvkehtv ttttkgenft etdikmmery  veqmcitqyq resqayyqrg asSEQ ID NO: 6 is an amino acid sequence of a recombinant ovine (136A 154R 171Q) proteinase K-sensitive prion protein. kkrpkp gggwntggsr ypgqgspggn ryppqggggw gqphgggwgq phgggwgqph gggwgqphgg ggwgqggshs qwnkpskpkt nmkhvagaaa agavvgglgg ymlgsamsrp lihfgndyed ryyrenmyry pnqvyyrpvd qysnqnnfvh dcvnitvkqh tvttttkgen ftetdikime rvveqmcitq  yqresqayyq rgaSEQ ID NO: 7 is an amino acid sequence of a recombinant Deer (96G 132M 138S) proteinase  K-sensitive prion protein.kkrpkp gggwntggsr ypgqgspggn ryppqggggw gqphgggwgq phgggwgqph gggwgqphgg ggwgqggths qwnkpskpkt nmkhvagaaa agavvgglgg ymlgsamsrp lihfgndyed ryyrenmyry pnqvyyrpvd qynnqntfvh dcvnitvkqh tvttttkgen ftetdikmme rvveqmcitq  yqresqayyq rgasSEQ ID NO: 8 is an amino acid sequence of a full-length Syrian golden hamster proteinase  K-sensitive prion protein.mwtdvglckk rpkpggwntg gsrypgqgsp ggnryppqgg gtwgqphggg wgqphgggwg qphgggwgqp hgggwgqggg thnqwnkpsk pktnmkhmag aaaagavvgg lggymlgsam srpmmhfgnd wedryyrenm nrypnqvyyr pvdqynnqnn fvhdcvniti kqhtvttttk genftetdik imervveqmc ttqyqkesqa yydgrrssav lfssppvill isfliflmvgSEQ ID NO: 9 is an amino acid sequence of a full-length mouse (Prnp-a) proteinase  K-sensitive prion protein.manlgywlla lfvtmwtdvg lckkrpkpgg wntggsrypg qgspggnryp pqggtwgqph gggwgqphgg swgqphggsw gqphgggwgq gggthnqwnk pskpktnlkh vagaaaagav vgglggymlg samsrpmihf gndwedryyr enmyrypnqv yyrpvdqysn qnnfvhdcvn itikqhtvtt ttkgenftet dvkmmervve qmcvtqyqke sqayydgrrs sstvlfsspp  villisflif livgSEQ ID NO: 10 is an amino acid sequence of a full-length human (129M) proteinase  K-sensitive prion protein.manlgewmlv lfvatwsdlg lckkrpkpgg wntggsrypg qgspggnryp pqggggwgqp hgggwgqphg ggwgqphggg wgqphgggwg qgggthsqwn kpskpktnmk hmagaaaaga vvgglggyml gsamsrpiih fgsdyedryy renmhrypnq vyyrpmdeys nqnnfvhdcv nitikqhtvt tttkgenfte tdvkmmervv eqmcitqyer esqayyqrgs smvlfssppv  illisflifl ivgSEQ ID NO: 11 is an amino acid sequence of a full-length human (129V) proteinase  K-sensitive prion protein.manlgewmlv lfvatwsdlg lckkrpkpgg wntggsrypg qgspggnryp pqggggwgqp hgggwgqphg ggwgqphggg wgqphgggwg qgggthsqwn kpskpktnmk hmagaaaaga vvgglggyvl gsamsrpiih fgsdyedryy renmhrypnq vyyrpmdeys nqnnfvhdcv nitikqhtvt tttkgenfte tdvkmmervv eqmcitqyer esqayyqrgs smvlfssppv  illisflifl ivg

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS I. Overview of SeveralEmbodiments

Disclosed herein is an ultrasensitive method, termed rPrp-resamplification, for detecting PrP^(Sc) that allows the use of purifiedrecombinant rPrP-sen as a substrate for seeded polymerization. Theresulting assay is much faster than previous PMCA methods, and the useof rPrP-sen facilitates improved prion assays and fundamental studies ofstructure and formation of PrP^(Sc). These methods can be used todiagnose a variety of diseases in animal and human subjects, and reducethe time necessary for high sensitivity detection of PrP-res in samples.Thus, the present disclosure also enables high throughput, accurate andsensitive screening of samples, as well as diagnosis of clinicaldisease.

In certain embodiments, the methods are used to diagnose a prion diseaseor a disease induced by a protein conformation change, such as aconformational change in PrP-sen. The disease can be a transmissiblespongiform encephalopathy, such as bovine spongiform encephalopathy(BSE) in a cow, whereas in sheep, the methods are used to diagnosescrapie, and in deer, elk, and moose the methods are used to diagnoseCWD. The method also enables the rapid testing of live animals forinfection to protect against unnecessary culling of herds or inadvertentintroduction of prions into the food chain.

The disclosed methods also are used to diagnose humans and humandiseases. Prion diseases that the methods detect in humans include butare not limited to Creutzfeldt-Jakob disease (CJD), kuru, fatal familialinsomnia, Gerstmann-Straussler-Scheinker disease, and sporadic fatalinsomnia. As when used for the diagnosis of animal diseases, thedisclosed methods offer significant advantages over available methodsfor diagnosis of these neurologic disorders. For instance, cognitivetests and clinical signs currently used for diagnosis of CJD can onlyindicate a probable diagnosis, and conventional PMCA takes up to threeweeks to perform, whereas the disclosed methods provide an objectivemethod by which positive diagnosis can be made within 1-2 days withlittle chance of false positive or false negative results. Additionally,the sensitivity of the test enables the detection of disease fromperipheral tissues, such as blood and cerebral spinal fluid (CSF), whichis much less invasive and expensive than brain biopsy procedures. Themethods also provide sensitivity that is sufficiently high to detect ordiagnose disease prior to the onset of clinical symptoms.

One embodiment of the disclosure is a method for detecting PrP-res in asample. The method includes (a) mixing the sample with purified rPrP-sento make a reaction mix (b) performing a primary reaction that includes(i) incubating the reaction mix to permit the coaggregation of therPrP-sen with the PrP-res that may be present in the reaction mix; (ii)agitating any aggregates formed during step (i); and (iii) repeatingsteps (i) and (ii) one or more times. In step (i) of the primaryreaction, aggregation of the rPrP-sen with the PrP-res results in aconversion of the rPrP-sen to rPrP-res^((Sc)). These amplification stepsare then followed by (c) detecting rPrP-res^((Sc)) in the reaction mix,wherein detection of rPrP-res^((Sc)) in the reaction mix indicates thatPrP-res was present in the sample. In some examples, steps (b)(i) and(b)(ii) are repeated from about 1 to about 200 times. In other examples,serial amplification is performed by removing a portion of the reactionmix and incubating it with additional rPrP-sen.

The reaction can be carried out by maintaining incubation conditions toinhibit production of rPrP-res^((spon)) which in the past has competedwith the desired reaction and may have contributed to the conclusionthat cyclic amplification could not be carried out with rPrP-sen. Thedetailed description describes a number of ways to inhibit production ofrPrP-res^((spon)), for example by one or more of (a) agitating theaggregates by shaking the reaction mix without sonication; (b)incubating the reaction mix in 0.05% to 0.1% of a detergent; (c)incubating the reaction mix at 37° C.-60° C.; or (d) incubating thereaction mix for no more than 2, 4, 6, 8, 16 or 20 hours at higherreaction temperatures. In certain examples, any combination of (a)-(d)or all of them are used to inhibit rPrP-res^((spon)) production, suchthat the amount of rPrP-res^((spon)) is less than 20% (or even less than15% or 10%) of that of rPrP-res^((Sc)) generated (in reactions seededwith samples containing PrP-res). The detergent may be a mixture ofdetergents, such as a mixture of an anionic and nonionic detergent, suchas SDS and Triton X-100.

In certain embodiments, the method also includes the step of performinga serial amplification reaction before detecting rPrP-res^((Sc)) in thereaction mix, wherein performing the serial reaction includes removing aportion of the reaction mix and incubating it with additional rPrP-sen.In other embodiments, detecting the PrP-resincludes detectingrPrP-res^((Sc)) aggregates in the reaction mix. Still other embodimentsalso include digesting the reaction mix with proteinase K prior todetecting rPrP-res^((Sc)) in the reaction mix. In certain examples,detecting rPrP-res^((Sc)) includes using an antibody that specificallybinds to prion protein, for instance D13, R18, or R20 antibodies.

In some embodiments of the disclosure, the PrP-res includes mammalianprion protein, and in certain examples, the rPrP-sen includes adetectable label. In other embodiments, incubating the reaction mixincludes incubating the reaction mix at about 25 to 70° C., and inparticular examples incubating the reaction mix includes incubating thereaction mix at about 37 to 55° C., or 45 to 55° C. In some examples,incubating the reaction mix includes incubating the reaction mix betweenagitations for about 1 to about 180 minutes, and in particular examplesincubating the reaction mix includes incubating the reaction mix for 1min or for about 60 to about 120 minutes, such as about 60, about 70,about 80, about 90, about 100, about 110 or about 110 minutes, forexample about 70 to about 100 minutes. In other examples, the reactionmix can be incubated for about 1, about 2, about 5, about 10, about 20,about 30, about 40 minutes between agitations. The total reaction time,including agitation and incubation can be about 2 to about 48 hours,such as about 4, about 6, about 8, about 16, about 20, about 24, about36, about 42, or about 48 hours.

In some examples of rPrP-PMCA, agitating the aggregates includessonicating the reaction mix, and in some examples, agitating thereaction mix includes sonicating the reaction mix for about 1-120seconds, or in other examples, for about 40 seconds. In otherembodiments, termed QUIC, agitating the reaction mix includes shakingthe reaction mix for about 1-120 seconds, for instance for about 10 or60 seconds. The primary reaction includes, in some embodiments, (a)incubating the reaction mix for approximately 60 minutes; and (b)sonicating the reaction mix for approximately 40 seconds. In someexamples, steps (a) and (b) are repeated for approximately 1-48 hours.

In other examples, the reaction mixture further includes an ionic (suchas an anionic) and a nonionic detergent, such as SDS and TRITON®(TX)-100, for example, from about 0.05% to about 0.1% SDS and from about0.05% to about 0.1% TX-100. Other suitable non-ionic detergents includeBis(polyethylene glycol bis[imidazoyl carbonyl]), Decaethylene glycolmonododecyl ether, N-Decanoyl-N-methylglucamine, n-Decyla-D-glucopyranoside, Decyl b-D-maltopyranoside,n-Dodecanoyl-N-methylglucamide, n-Dodecyl a-D-maltoside, n-Dodecylb-D-maltoside, n-Dodecyl b-D-maltoside, SigmaUltra, Heptaethylene glycolmonodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethyleneglycol monotetradecyl ether, n-Hexadecyl b-D-maltoside, Hexaethyleneglycol monododecyl ether, Hexaethylene glycol monohexadecyl ether,Hexaethylene glycol monooctadecyl ether, Hexaethylene glycolmonotetradecyl ether,Methyl-6-O-(N-heptylcarbamoyl)-α-D-glucopyranoside, Nonaethylene glycolmonododecyl ether, N-Nonanoyl-N-methylglucamine,N-Nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether,Octaethylene glycol monododecyl ether, Octaethylene glycol monohexadecylether, Octaethylene glycol monooctadecyl ether, Octaethylene glycolmonotetradecyl ether, Octyl-b-D-glucopyranoside, Pentaethylene glycolmonodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethyleneglycol monohexadecyl ether, Pentaethylene glycol monohexyl ether,Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctylether, Polyethylene glycol diglycidyl ether, Polyethylene glycol etherW-1, Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate,Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether,Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate,Polyoxyethylene 8 stearate, Polyoxyethylene bis(imidazolyl carbonyl),Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillaj abark, SPAN® (Nos. 20, 40, 60, 65 80, or 85), Tergitol (Type 15-S-12,Type 15-S-30, Type 15-S-5, Type 15-S-7, Type 15-S-9, Type NP-10, TypeNP-4, Type NP-40, Type NP-7, Type NP-9, MIN FOAM lx, MIN FOAM 2×, TypeTMN-10, Type TMN-6), Tetradecyl-b-D-maltoside, Tetraethylene glycolmonodecyl ether, Tetraethylene glycol monododecyl ether, Tetraethyleneglycol monotetradecyl ether, Triethylene glycol monodecyl ether,Triethylene glycol monododecyl ether, Triethylene glycol monohexadecylether, Triethylene glycol monooctyl ether, Triethylene glycolmonotetradecyl ether, TRITON® CF-21, TRITON® CF-32, TRITON® DF-12,TRITON® DF-16, TRITON® GR-5M, Triton X-100, Triton X-102, TRITON® X-15,TRITON® X-151, TRITON® X-207, TRITON® X-100, TRITON® X-114 TRITON®X-165, TRITON® X-305, TRITON® X-405, TRITON® X-45, TRITON® X-705-70,TWEEN® 20, TWEEN® 21, TWEEN® 40, TWEEN® 60, TWEEN® 6, TWEEN® 65, TWEEN®80, TWEEN® 81, TWEEN® 85, Tyloxapol, and n-Undecyl b-D-glucopyranoside.Other suitable anionic detergents of use include Chenodeoxycholic acid,Chenodeoxycholic acid sodium salt, Cholic acid, ox or sheep bile,Dehydrocholic acid, Deoxycholic acid, Deoxycholic acid methyl ester,Digitonin, Digitoxigenin, N,N-Dimethyldodecylamine N-oxide, Docusatesodium salt, Glycochenodeoxycholic acid sodium salt, Glycocholic acidhydrate (Glycodeoxycholic acid monohydrate, Glycodeoxycholic acid sodiumsalt, Glycolithocholic acid 3-sulfate disodium salt, Glycolithocholicacid ethyl ester), N-Lauroylsarcosine, Lithium dodecyl sulfate, Niaproof4, TRITON® QS-15, TRITON® QS-44, 1-Octanesulfonic acid sodium salt,Sodium 1-butanesulfonate, Sodium 1-decanesulfonate, Sodium1-dodecanesulfonate, Sodium 1-heptanesulfonate anhydrous, Sodium1-nonanesulfonate, Sodium 1-propanesulfonate monohydrate, Sodium2-bromoethanesulfonate, Sodium cholate hydrate, Sodium choleate, Sodiumdeoxycholate, Sodium deoxycholate monohydrate, Sodium hexanesulfonate,Sodium octyl sulfate, Sodium pentanesulfonate, Sodium taurocholate,Taurochenodeoxycholic acid sodium salt, Taurodeoxycholic acid sodiumsalt monohydrate, Taurodeoxycholic acid sodium salt monohydrate,Taurohyodeoxycholic acid sodium salt hydrate, Taurolithocholic acid3-sulfate disodium salt Tauroursodeoxycholic acid sodium salt, TRITON®X-200M TRITON® XQS-20, TRIZMA® dodecyl sulfate, and Ursodeoxycholicacid. The anionic and inonic detergents can be used for example, at aconcentration of 0.01% to 0.5%, such as 0.05% to 0.1%.

In some embodiments, the source of the recombinant rPrP-sen is the samespecies as the source of the sample or it is of a different species asthe source of the sample. It has particularly been found that rHaPrP-senis well suited to the amplification reaction, and can be used to amplifytarget protein in a target from species other than hamster. Someexamples of the method include a rPrP-sen that is bovine, ovine,hamster, rat, mouse, canine, feline, cervid, human, or non-human primaterPrP-sen. In certain examples, the rPrP-sen includes amino acids 23-231(SEQ ID NO: 1) of Syrian golden hamster prion protein (SEQ ID NO: 8),amino acids 23-231 (SEQ ID NO: 2) of mouse prion protein (SEQ ID NO: 9),amino acids 23-231 (SEQ ID NO: 3) of human (129M) prion protein (SEQ IDNO: 10), amino acids 23-231 (SEQ ID NO: 4) of human (129V) prion protein(SEQ ID NO: 11), amino acids 25-241 (SEQ ID NO: 5) of bovine(6-octarepeat) prion protein, amino acids 25-233 (SEQ ID NO: 6) of ovine(136A 154R 171Q) prion protein, or amino acids 25-234 (SEQ ID NO: 7) ofdeer (96G 132M 138S) prion protein. However, fragments of rPrP-sen canalso be used, such as but not limited to a fragment comprising aminoacids 23-231 of rPrP-sen. Fragments include amino acids 30-231, aminoacids 40-231, amino acids 50-231, amino acids 60-231, amino acids70-231, amino acids 80-231 or amino acids 90-231 of mouse, human,hamster, bovine, ovine or deer prion protein. A functional fragment ofrPrP-sen can aggregate with PrP-res and result in a conversion of therPrP-sen to rPrP-res^((Sc)). It should be noted that chimeric rPrP-sen,wherein a portion of the protein is from one species, and a portion ofthe protein is from another species, can also be utilized. In oneexample about 10 to about 90%, such as about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70% about 80% or about 90% of therPrP-sen is from one species, and, correspondingly, about 90%, about80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20% orabout 10% is from another species. Chimeric proteins can include, forexample, hamster rPrP-sen and rPrP-sen from another species, such ashuman PrP-sen.

Particular examples include a sample that is a tissue sample from ananimal, for instance a brain sample, a peripheral organ sample, feces,urine, mucosal secretions, or a CSF sample. In even more particularexamples, the peripheral organ sample includes blood, tonsil, nasaltissue, spleen, or another lymphoid organ.

Detecting PrP-res^((Sc)) in the reaction mix includes, in someembodiments, performing a Western blot, an ELISA assay, a CDI assay, aDELPHIA assay, a strip immuno-chromatographic assay, a spectroscopicassay, a fluorescence assay, or a radiometric assay. In certainexamples, the ELISA assay is a two-site immunometric sandwich ELISA. Inparticular examples, the prion can be detected in a sample containing atleast 1000 PrP^(Sc) molecules. In still more particular examples, themethod further includes inactivating residual PrP-res in the reactionmix. In yet other examples, the method is a method of diagnosing a priondisease.

Also disclosed herein are kits for detection of a prion in a sample thatinclude rPrP-sen and at a reaction mix buffer. In some embodiments, thereaction mix buffer includes SDS and TX-100, and in other embodiments,the rPrP-sen is lyophilized. In certain examples, the kit also includesone or more of (a) a decontamination solution; (b) a positive control;(c) a negative control; or (d) reagents for the detection ofrPrP-res^((Sc)). In particular examples, the reagents for detection ofrPrP-res^((Sc)) include antibodies.

In yet other embodiments, a rPrP-res amplification method is disclosedfor detecting PrP-res in a sample by

(a) mixing the sample with purified rPrP-sen to make a reaction mix; andperforming an amplification reaction in a single round without serialamplification, by incubating the reaction mixture to permitcoaggregation of the rPrP-sen with the PrP-res that may be present inthe reaction mix, wherein coaggregation of the rPrP-sen with the PrP-resresults in a conversion of the rPrP-sen to the rPrP-res^((Sc)). Usingthis method it is possible to sensitively detect PrP-res in a sampleunder a variety of conditions, such as conditions (b) through (e) below:

(b) detecting PrP-res in a sample containing as little as 100 ag PrP-resby incubating the reaction mixture at 45° C. for about 46 hours andintermittently shaking the reaction mixture without sonication, andrPrP-res^((Sc)) is detected as an indicator of the initial presence ofPrP-res;

(c) detecting PrP-res in a sample containing as little as 1 fg PrP-resby incubating the reaction mixture at 55° C. for about 18 hours andintermittently shaking the reaction mixture without sonication, andrPrP-res^((Sc)) is detected as an indicator of the initial presence ofPrP-res;

(d) detecting PrP-res in a sample containing as little as 10 fg PrP-resby incubating the reaction mixture at 55° C. for about 8 hours andintermittently shaking the reaction mixture without sonication, andrPrP-res^((Sc)) is detected as an indicator of the initial presence ofPrP-res; and

(e) detecting PrP-res in a sample containing as little as 100 fg PrP-resby incubating the reaction mixture at 65° C. for about 4 hours andintermittently shaking the reaction mixture without sonication, andrPrP-res^((Sc)) is detected as an indicator of the initial presence ofPrP-res.

II. Abbreviations

BH: brain homogenate

BSE: bovine spongiform encephalopathy

CJD: Creutzfeldt-Jakob disease

CSF: cerebral spinal fluid

CWD: chronic wasting disease

EEG: electroencephalogram

ELISA: enzyme linked immunosorbent assays

EUE: exotic ungulate encephalopathy

fCJD: familial Creutzfeldt-Jakob disease

FFI: fatal familial insomnia

GFP: green fluorescent protein

GSS: Gerstmann-Strussler Sheinker syndrome

GST: Glutathione S-transferase

HaPrP-res: hamster proteinase K resistant prion protein

HaPrP^(Sc): hamster proteinase K resistant prion protein

HaPrP-sen: hamster proteinase K sensitive prion protein

iCJD: iatrogenic Creutzfeldt-Jakob disease

MBP: Maltose binding protein

NBH: normal brain homogenate

PBS: phosphate buffered saline

PK: proteinase K

PMCA: protein misfolding cyclic amplification

PrP-res: proteinase K resistant prion protein

PrP^(Sc): proteinase K resistant prion protein

PrP-sen: proteinase K sensitive prion protein

QUIC: quaking-induced conversion

RIA: radioimmunoassay

rHaPrP-res^((vCJD)):recombinant hamseter proteinase K resistant prionprotein, variant Creutzfeldt-Jakob disease that arises from seedinghamster PrP-res into a human sample

rPrP-res: recombinant proteinase K resistant prion protein

rPrP-res^((Sc)): recombinant proteinase K resistant prion protein seededby PrP^(Sc)

rPrP-res^((spon)): recombinant proteinase K resistant prion protein thatspontaneously arises without seeding (unseeded) by rPrP-res

rPrP-sen: recombinant proteinase K sensitive prion protein

ScBH: Scrapie brain homogenate

sCJD: sporadic Creutzfeldt-Jakob disease

SDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis

sFI: sporadic fatal insomnia

TSE: transmissible spongiform encephalopathy

TX-100: Triton X-100

vCJD: variant Creutzfeldt-Jakob disease

III. Terms

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology can be found in Benjamin Lewin, Genes V,published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrewet al. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. The term “plurality” refers to two or more. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described herein. The term “comprises” means “includes.”

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Aggregate: as used herein, includes aggregates, dimers, multimers, andpolymers of prion proteins, for instance aggregates, dimers, multimers,and polymers of PrP-res, rPrP-res, or rPrP-res^((Sc))

Agitation: includes introducing any type of turbulence or motion into amixture or reaction mix, for examples by sonication, stiffing, orshaking. In some embodiments, agitation includes the use of forcesufficient to fragment rPrP-res^((Sc)) aggregates, which dispersesrPrP-res^((Sc)) aggregates and/or polymers to facilitate furtheramplification. In some examples fragmentation includes completefragmentation, whereas in other examples, fragmentation is only partial,for instance, a population of aggregates can be about 1%, 2%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% fragmented by agitation.Exemplary agitation methods are described in the Examples section below.

Conservative variant: in the context of a prion protein, refers to apeptide or amino acid sequence that deviates from another amino acidsequence only in the substitution of one or several amino acids foramino acids having similar biochemical properties (so-calledconservative substitutions). Conservative amino acid substitutions arelikely to have minimal impact on the activity of the resultant protein.Further information about conservative substitutions can be found, forinstance, in Ben Bassat et al. (J. Bacteriol., 169:751-757, 1987),O'Regan et al. (Gene, 77:237-251, 1989), Sahin-Toth et al. (ProteinSci., 3:240-247, 1994), Hochuli et al. (Bio/Technology, 6:1321-1325,1988) and in widely used textbooks of genetics and molecular biology. Insome examples, prion protein variants can have no more than 1, 2, 3, 4,5, 10, 15, 30, 45, or more conservative amino acid changes. Conservativevariants are discussed in greater detail in section IV F of the DetailedDescription.

In one example, a conservative variant prion protein is one thatfunctionally performs substantially like a similar base component, forinstance, a prion protein having variations in the sequence as comparedto a reference prion protein. For example, a prion protein or aconservative variant of that prion protein, will aggregate with rPrP-sen(or PrP^(Sc)), for instance, and will convert rPrP-sen to rPrP-res (orwill be converted to rPrP-res). In this example, the prion protein andthe conservative variant prion protein do not have the same amino acidsequences. The conservative variant can have, for instance, onevariation, two variations, three variations, four variations, or five ormore variations in sequence, as long as the conservative variant isstill complementary to the corresponding prion protein.

In some embodiments, a conservative variant prion protein includes oneor more conservative amino acid substitutions compared to the prionprotein from which it was derived, and yet retains prion proteinbiological activity. For example, a conservative variant prion proteincan retain at least 10% of the biological activity of the parent prionprotein molecule from which it was derived, or alternatively, at least20%, at least 30%, or at least 40%. In some preferred embodiments, aconservative variant prion protein retains at least 50% of thebiological activity of the parent prion protein molecule from which itwas derived. The conservative amino acid substitutions of a conservativevariant prion protein can occur in any domain of the prion protein.

Disaggregate: To partially or complete disrupt an aggregate, such as anaggregate of PrP-res, rPrP-res, or rPrP-res^((Sc)).

Encode: any process whereby the information in a polymeric macromoleculeor sequence is used to direct the production of a second molecule orsequence that is different from the first molecule or sequence. As usedherein, the term is construed broadly, and can have a variety ofapplications. In some aspects, the term “encode” describes the processof semi-conservative DNA replication, wherein one strand of adouble-stranded DNA molecule is used as a template to encode a newlysynthesized complementary sister strand by a DNA-dependent DNApolymerase.

In another aspect, the term “encode” refers to any process whereby theinformation in one molecule is used to direct the production of a secondmolecule that has a different chemical nature from the first molecule.For example, a DNA molecule can encode an RNA molecule (for instance, bythe process of transcription incorporating a DNA-dependent RNApolymerase enzyme). Also, an RNA molecule can encode a peptide, as inthe process of translation. When used to describe the process oftranslation, the term “encode” also extends to the triplet codon thatencodes an amino acid. In some aspects, an RNA molecule can encode a DNAmolecule, for instance, by the process of reverse transcriptionincorporating an RNA-dependent DNA polymerase. In another aspect, a DNAmolecule can encode a peptide, where it is understood that “encode” asused in that case incorporates both the processes of transcription andtranslation.

Hybridization: Oligonucleotides and their analogs hybridize by hydrogenbonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases. Generally, nucleic acidconsists of nitrogenous bases that are either pyrimidines (cytosine (C),uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)).These nitrogenous bases form hydrogen bonds between a pyrimidine and apurine, and the bonding of the pyrimidine to the purine is referred toas “base pairing.” More specifically, A will hydrogen bond to T or U,and G will bond to C. “Complementary” refers to the base pairing thatoccurs between two distinct nucleic acid sequences or two distinctregions of the same nucleic acid sequence. For example, anoligonucleotide can be complementary to a prion protein-encoding RNA, ora prion protein-encoding DNA.

“Specifically hybridizable” and “specifically complementary” are termsthat indicate a sufficient degree of complementarity such that stableand specific binding occurs between the oligonucleotide (or its analog)and the DNA or RNA target. The oligonucleotide or oligonucleotide analogneed not be 100% complementary to its target sequence to be specificallyhybridizable. An oligonucleotide or analog is specifically hybridizablewhen binding of the oligonucleotide or analog to the target DNA or RNAmolecule interferes with the normal function of the target DNA or RNA,and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide or analog to non-targetsequences under conditions where specific binding is desired, forexample under physiological conditions in the case of in vivo assays orsystems. Such binding is referred to as specific hybridization.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na+ and/or Mg++ concentration) of thehybridization buffer will determine the stringency of hybridization,though wash times also influence stringency. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed by Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11.

In a particular example, stringent conditions are hybridization at 65°C. in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg sheared salmontestes DNA, followed by 15 30-minute sequential washes at 65° C. in2×SSC, 0.5% SDS, followed by 1×SSC, 0.5% SDS and finally 0.2×SSC, 0.5%SDS.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, peptide, or cell) has been purified away from other biologicalcomponents in a mixed sample (such as a cell extract). For example, an“isolated” peptide or nucleic acid molecule is a peptide or nucleic acidmolecule that has been separated from the other components of a cell inwhich the peptide or nucleic acid molecule was present (such as anexpression host cell for a recombinant peptide or nucleic acidmolecule).

Nucleic acid molecule: A polymeric form of nucleotides, which caninclude both sense and anti sense strands of RNA, cDNA, genomic DNA, andsynthetic forms and mixed polymers of the above. A nucleotide refers toa ribonucleotide, deoxynucleotide or a modified form of either type ofnucleotide. A “nucleic acid molecule” as used herein is synonymous with“nucleic acid” and “polynucleotide.” A nucleic acid molecule is usuallyat least 10 bases in length, unless otherwise specified. The termincludes single and double stranded forms of DNA. A nucleic acidmolecule can include either or both naturally occurring and modifiednucleotides linked together by naturally occurring and/or non naturallyoccurring nucleotide linkages.

Prion: a type of infectious agent composed mainly of protein. Prionscause a number of diseases in a variety of animals, including bovinespongiform encephalopathy (BSE, also known as mad cow disease) in cattleand Creutzfeldt-Jakob disease in humans. All known prion diseases affectthe structure of the brain or other neural tissue, and all areuntreatable and fatal.

Prions are believed to infect and propagate by refolding abnormally intoa structure that is able to convert normal molecules of the protein intothe abnormally structured (for instance, PrP-res or Prp^(Sc)) form.Most, if not all, known prions can polymerize into amyloid fibrils richin tightly packed beta sheets. This altered structure renders themunusually resistant to denaturation by chemical and physical agents,making disposal and containment of these particles difficult.

In prion diseases, the pathological, protease-resistant form of prionprotein, termed PrP^(Sc) or PrP-res, appears to propagate itself ininfected hosts by inducing the conversion of its normal host-encodedprotease-sensitive precursor, PrP-sen, into PrP^(Sc). PrP-sen is amonomeric glycophosphatidylinositol-linked glycoprotein that is low inβ-sheet content, and highly protease-sensitive. Conversely, PrP^(Sc)aggregates are high in β-sheet content and partially protease-resistant.Mechanistic details of the conversion are not well understood, butinvolve direct interaction between PrP^(Sc) and PrP-sen, resulting inconformational changes in PrP-sen as the latter is recruited into thegrowing PrP^(Sc) multimer (reviewed in Caughey & Baron (2006) Nature443, 803-810). Accordingly, the conversion mechanism has beententatively described as autocatalytic seeded (or nucleated)polymerization.

PMCA or Protein Misfolding Cyclic Amplification: A method for amplifyingPrP-res in a sample by mixing Prp-sen with the sample, incubating thereaction mix to permit PrP-res to initiate the conversion of PrP-sen toaggregates of PrP-res, fragmenting any aggregates formed during theincubation step (typically by sonication), and repeating one or morecycles of the incubation and fragmentation steps.

QUIC or Quaking Induced Conversion: A particular type of rPrP-senamplification assay, in which shaking of the reaction vessels isperformed instead of sonication to disrupted aggregated PrP-sen andPrP-res.

Sequence identity: The similarity between two nucleic acid sequences orbetween two amino acid sequences is expressed in terms of the level ofsequence identity shared between the sequences. Sequence identity istypically expressed in terms of percentage identity; the higher thepercentage, the more similar the two sequences. Methods for aligningsequences for comparison are described in detail below, in section IV Eof the Detailed Description.

Single Round: Performing a method wherein serial amplification is notperformed. For example, PrP-res can be amplified in a sample, by mixingthe sample with purified rPrP-sen to make a reaction mix; performing anamplification reaction that includes (i) incubating the reaction mix topermit coaggregation of the rPrP-sen with the PrP-res that may bepresent in the reaction mix, and maintaining incubation conditions thatpromote coaggregation of the rPrP-sen with the PrP-res and results in aconversion of the rPrP-sen to rPrP-res^((Sc)) while inhibitingdevelopment of rPrP-res^((spon)); (ii) agitating aggregates formedduring step (i); (iii) optionally repeating steps (i) and (ii) one ormore times. rPrP-res^((Sc)) is detected in the reaction mix, whereindetection of rPrP-res^((Sc)) in the reaction mix indicates that PrP-reswas present in the sample. However, a portion of the reaction mix is notremoved and incubated with additional rPrP-sen.

Suitable methods and materials for the practice or testing of thedisclosure are described below. However, the provided materials,methods, and examples are illustrative only and are not intended to belimiting. Accordingly, except as otherwise noted, the methods andtechniques of the present disclosure can be performed according tomethods and materials similar or equivalent to those described and/oraccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification (see, for instance,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, 1989; Sambrook et al., MolecularCloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001;Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates, 1992 (and Supplements to 2000); Ausubel et al.,Short Protocols in Molecular Biology: A Compendium of Methods fromCurrent Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999).

IV. Detailed Description of Particular Embodiments A. Overview of Prionsand Prion Disease

The transmissible spongiform encephalopathies (TSEs, or prion diseases)are infectious neurodegenerative diseases of mammals that include (butare not limited to) scrapie in sheep, bovine spongiform encephalopathy(BSE; also known as mad cow disease) in cattle, transmissible minkencephalopathy (TME) in mink, chronic wasting disease (CWD) in elk,moose, and deer, feline spongiform encephalopathy in cats, exoticungulate encephalopathy (EUE) in nyala, oryx and greater kudu, andCreutzfeldt-Jakob disease (CJD) and its varieties (iatrogenicCreutzfeldt-Jakob disease (iCJD), variant Creutzfeldt-Jakob disease(vCJD), familial Creutzfeldt-Jakob disease (fCJD), and sporadicCreutzfeldt-Jakob disease (sCJD)), Gerstmann-Strässler-Scheinkersyndrome (GSS), fatal familial insomnia (fFI), sporadic fatal insomnia(sFI), kuru, and Alpers syndrome in humans. TSEs have incubation periodsof months to years, but after the appearance of clinical signs often arerapidly progressive, untreatable, and invariably fatal. Attempts at TSErisk reduction have led to profound changes in the production and tradeof agricultural goods, medicines, cosmetics, and biotechnology products.

In TSEs the pathological, protease-resistant form of prion protein,termed PrP^(Sc) or PrP-res, appears to propagate itself in infectedhosts by inducing the conversion of its normal host-encoded precursor,PrP-sen, into PrP^(Sc). PrP-sen is a monomericglycophosphatidylinositol-linked glycoprotein that is low in β-sheetcontent, and highly protease-sensitive. Conversely, PrP^(Sc) aggregatesare high in β-sheet content and partially protease-resistant.Mechanistic details of the conversion are not well understood, butinvolve direct interaction between PrP^(Sc) and PrP-sen, resulting inconformational changes in PrP-sen as the latter is recruited into thegrowing PrP^(Sc) multimer (reviewed in Caughey & Baron (2006) Nature443, 803-810). Accordingly, the conversion mechanism has beententatively described as autocatalytic seeded (or nucleated)polymerization.

To better understand the mechanism of prion propagation, many attemptsto recapitulate PrP^(Sc) formation in cell-free systems have been made.Initial experiments showed that PrP^(Sc) can induce the conversion ofPrP-sen to PrP^(Sc) with strain- and species-specificities, albeit withsubstoichiometric yields. More recently, it was shown that PrP^(Sc)formation and TSE infectivity can be amplified indefinitely in crudebrain homogenates, a medium containing numerous potential cofactors forconversion (Castilla et al., (2005) Cell 121, 195-206). Dissection ofthis “protein misfolding cyclic amplification” (PMCA) reaction showedthat PrP^(Sc) and prion infectivity also could be amplified usingPrP-sen purified from brain tissue as long as polyanions such as RNAwere added (Deleault et al., (2007) Proc Natl Acad Sci USA.104(23):9741-6). Recombinant PrP-sen (rPrP-sen) from E. coli lacksglycosylation and the GPI anchor and prior to this disclosure has notbeen used successfully as an amplification substrate in PrP^(Sc)-seededPMCA reactions. In fact, it was previously reported that rPrP-sen doesnot work in the PMCA system (Nishina et al., (2006) Biochemistry45(47):14129-39). However, rPrP-sen can be converted toprotease-resistant forms with limited yields when mixed with PrP^(Sc).rPrP-sen also can be induced to polymerize into amyloid fibrilsspontaneously or when seeded by preformed rPrP fibrils. Although mostrPrP amyloid preparations are not infectious, synthetic amyloid fibrilsof mutant recombinant prion protein can cause or accelerate TSE diseasein transgenic mice that vastly overexpress the same mutant prion proteinconstruct (Legname et al. (2004) Science 305, 673-676). However, these“synthetic prions” were non-infectious for wild type mice, making themat least 10⁸-fold less infectious than bona fide PrP^(Sc). Thus, thebasic structure and propagation mechanism of robust TSE infectivity (orprions) remains to be fully ascertained.

A key challenge in coping with TSEs is the rapid detection of low levelsof TSE infectivity (prions) by rapid methods. The most commonly usedmarker for TSE infections is PrP^(Sc), and the PMCA reaction allowsextremely sensitive detection of PrP^(Sc) at levels below singleinfectious units in infected tissue. However, as previously noted,current limitations of PMCA include the time required to achieve optimalsensitivity (˜3 weeks) and the use of brain PrP-sen as the amplificationsubstrate.

B. Transmissible Spongiform Encephalopathies (TSEs)

The most common TSE in animals is scrapie, but the most famous anddangerous TSE is BSE, which affects cattle and is known by its lay term“mad cow disease.” In humans, the most common TSE is CJD, which occursworldwide with an incidence of 0.5 to 1.5 new cases per one millionpeople each year. Three different forms of CJD have been traditionallyrecognized: sporadic (sCJD; 85% of cases), familial (fCJD; 10%), andiatrogenic (iCJD; 5%). However, in 1996, a new variant form of CJD(vCJD) emerged in the UK that was associated with consumption of meatinfected with BSE. In contrast with typical sCJD, vCJD affects youngpatients with an average age of 27 years, and causes a relatively longillness (14 months compared with 4.5 months for sCJD). Because ofinsufficient information available about the incubation time and thelevels of exposure to contaminated cattle food products, it is difficultto predict the future incidence of vCJD. In animals, there is noevidence for inherited forms of the disease, and most cases appear to beacquired by horizontal or vertical transmission.

The clinical diagnosis of sCJD is based on a combination of rapidlyprogressive multifocal dementia with pyramidal and extrapyramidal signs,myoclonus, and visual or cerebellar signs, associated with acharacteristic periodic electroencephalogram (EEG). A key diagnosticfeature of sCJD that distinguishes it from Alzheimer's disease and otherdementias is the rapid progression of clinical symptoms and the shortduration of the disease, which is often less than 2 years. The clinicalmanifestation of fCJD is very similar, except that the disease onset isslightly earlier than in sCJD. Family history of inherited CJD orgenetic screening for mutations in the prion protein gene are used toestablish fCJD diagnosis, although lack of family history does notexcludes an inherited origin.

Variant CJD appears initially as a progressive neuropsychiatric disordercharacterized by symptoms of anxiety, depression, apathy, withdrawal anddelusions, combined with persistent painful sensory symptoms andfollowed by ataxia, myoclonus, and dementia. Variant CJD isdifferentiated from sCJD by the duration of illness (usually longer than6 months) and EEG analysis (vCJD does not show the atypical patternobserved in sCJD). A high bilateral pulvinar signal noted during MRI isoften used to help diagnose vCJD. In addition, a tonsil biopsy can beused to help diagnose vCJD, based on a number of cases of vCJD have beenshown to test positive for PrPSc staining in lymphoid tissue (such astonsil and appendix). However, because of the invasive nature of thistest, it is performed only in patients who fulfill the clinical criteriaof vCJD where the MRI of the brain does not show the characteristicpulvinar sign.

GSS is a dominantly inherited illness that is characterized by dementia,Parkinsonian symptoms, and a relatively long duration (typically, 5-8years). Clinically, GSS is similar to Alzheimer's disease, except thatis often accompanied by ataxia and seizures. Diagnosis is established byclinical examination and genetic screening for prion protein mutations.FFI is also dominantly inherited and associated with prion proteinmutations. However, the major clinical finding associated with EH isinsomnia, followed at late stages by myoclonus, hallucinations, ataxia,and dementia.

C. Protein Misfolding Cyclic Amplification (PMCA), and rPrP-ResAmplification (rPrP-PMCA and QUIC)

The prion detection method termed protein misfolding cyclicamplification (PMCA) is based on the ability of prions to replicate invitro in tissue homogenates containing PrP-sen (see, for instance,WO0204954). PMCA involves amplification of a PrP-res through incubationwith a suitable prion protein substrate derived from brain tissue,serial amplification of the PrP-res, for instance by alternatingincubation and sonication steps, and detection of the resultingPrP-res^((Sc)). In some instances, incubation and sonication arealternated over a period of approximately three weeks, andintermittently a portion of the reaction mix is removed and incubatedwith additional PrP-sen in order to serially amplify the PrP-res in thesample. Following the repeated incubation/sonication/dilution steps, theresulting PrP-res^((Sc)) is detected in the reaction mix. Although PMCAis a very sensitive assay for detecting PrP-res, it has a number oflimitations, notably the time required to achieve optimal sensitivity(−3 weeks) and the requirement for brain-derived PrP-sen as theamplification substrate.

The development of more sensitive, rapid, and practical means fordetection of PrP^(Sc) and TSE infectivity is critical in addressing thechallenges posed by prion diseases. Such a test could be used toidentify sources of TSE infection in agriculture and the environment toreduce risks to humans and animals. Moreover, the ability to diagnoseinfections in humans long before the appearance of clinical signs wouldgreatly improve the chances of treating these otherwise fatal diseases.Indeed, drug treatments in animals tend to be much more effective whentreatments are initiated within the first two thirds of the incubationperiod Caughey et al. (2006) Accts. Chem. Res. 39, 646-653; Trevitt &Collinge (2006) Brain 129, 2241-2265).

Disclosed herein is an improved prion assay, termed rPrP-resamplification assay (including rPrP-PMCA and QUIC), that differs fromthe PMCA PrP^(Sc) amplification method (Saa et al., (2006) J. Biol.Chem. 281, 35245-35252; Saa et al., (2006) Science 313, 92-94).rPrP-PMCA greatly improves the practicality of the basic PMCA approachin several significant ways. First, instead of prion protein substratederived from brain tissue, rPrP-PMCA and QUIC (when agitation isperformed by shaking) makes use of bacterially-expressed rPrP-sen as asubstrate, which can be obtained rapidly in high purity and in largeamounts, whereas purification of PrP-sen from brain tissue is difficultand gives much lower yields (Deleault et al. (2005) J. Biol. Chem. 280,26873-26879; Pan et al. (1993) Proc. Natl. Acad. Sci. USA 90,10962-10966; Hornemann et al., (2004) EMBO Rep. 5, 1159-1164).Furthermore, unlike PrP-sen in brain homogenates or purified from brain,rPrP-sen can be easily mutated or strategically labeled with probes tosimplify and accelerate the detection of relevant rPrP-PMCA products.

There are two types of rPrP-res amplification methods that utilizerPrP-sen, one that uses sonication (rPrP-PMCA) and one that utilizesshaking (QUIC). These methods facilitate fundamental studies of thestructure and conversion mechanism of PrP^(Sc). Site-directed mutationscan allow precise labeling of rPrP-sen with a variety of probes that canreport on conformational changes, and both inter-molecular andintra-molecular distances within rPrP-res aggregates.

The rPrP-PMCA and QUIC methods generally involve mixing a sample (forexample a tissue sample or CSF sample that is suspected of containingPrP-res) with purified rPrP-sen to make a reaction mix, and performing aprimary reaction to form and amplify specific forms of rPrP-res in themixture. This primary reaction includes incubating the reaction mix topermit the PrP-res to initiate the conversion of rPrP-sen to specificaggregates or polymers of rPrP-res; fragmenting any aggregates orpolymers formed during the incubation step; and repeating the incubationand fragmentation steps one or more times, for instance from about 10 toabout 50 times. In some embodiments of the method, serial amplificationis carried out by removing a portion of the reaction mix and incubatingit with additional rPrP-sen. Following amplification, theprion-initiated rPrP-res^((Sc)) in the reaction mix is detected, forexample using an antibody. In some examples, the reaction mix isdigested with proteinase K (which digests the remaining rPrP-sen in thereaction mix) prior to detection of the rPrP-res^((Sc)). Two types ofmis-folded prion protein can be generated in rPrP-PMCA (or QUIC)reactions, one occurring spontaneously (rPrP-res^((spon))) and the otherinitiated by the presence of prions (rPrP-res^((Sc)) in the test sample.Thus, it is often necessary to discriminate between the former and thelatter to interpret the rPrP-PMCA assay. For instance, this can be doneon the basis of differing protein fragment sizes generated upon exposureto proteinase K. An unexpectedly superior decrease in the amount ofrPrP-res^((spon))) formed is achieved with the QUIC assay.

The use of recombinant prion protein as a substrate for the QUICreaction instead of PrP-sen contained in, or isolated from, brainhomogenates (which is the source of substrate in conventional PMCA)confers several advantages. For instance, successful expression andfolding of rPrP enables the generation of large amounts of highlypurified and concentrated substrate, which is not possible when the onlyavailable source of substrate is brain tissue. Additionally andsurprisingly, the use of concentrated recombinant prion protein promotesfar faster amplification reactions than does PrP-sen in brainhomogenate. It is this surprising functionality of the rPrP that reducesthe time required for the reaction from up to three weeks to about 1-2days, or even less than a day.

All of the methods disclosed herein, such as QUIC, will work under avariety of conditions. In several embodiments, optimal conditions thatsupport specific PrP^(Sc)-seeded QUIC include the use of a detergent,such as both an ionic and a non-ionic detergent. The conditions caninclude the combination of about 0.05-0.1% of an ionic detergent such asSDS and about 0.05-0.1% of a nonionic detergent such as TX-100 in thereaction mix. Other preferred conditions include the use of shakinginstead of sonication (the so-called QUIC reaction), and the use ofcycles of shaking/rest that are about 1:1 in duration. Reactions havealso been found to be particularly efficient at 37-60° C., for example45-55° C. These conditions are particularly effective at promoting theformation of rPrP-res^((Sc)) (notably the 17 kDa PK-resistant species),while reducing rPrP-res^((spon)) formation within the first 24 hours ofunseeded reactions. However, longer amplification reactions of more than24 hours, such as at least 45 hours or even 65 or 96 hours, can alsoprovide excellent results.

The sensitivity of the assay has been found to be degraded (andpotential false positive results are obtained) by the production ofrPrP-res^((spon)) in the use of rPrP-sen seeded reactions. To help avoidthis problem, conditions are selected to inhibit the formation of therPrP-res^((spon)) byproduct. In some examples, assays (such as QUIC) areperformed to test assay conditions to determine if the assay conditionsincrease or decrease rPrP-res^((spon)) byproduct formation, and assayconditions are selected that minimize the byproduct formation. Therecognition of this previously unappreciated obstacle to the use ofamplification assays has also helped provide a much faster and moresensitive assay to address this substantial public health concern.

The sensitivity achieved with rPrP-PMCA (and QUIC) is of considerableutility because it is very sensitive. For example, the assay allowsconsistent detection of HaPrP^(Sc) levels (50 ag) that are >100-foldlower than those typically associated with a lethal intracerebral doseof 263K strain scrapie infectivity in Syrian golden hamsters. Althoughthis detection limit is not quite as low as that reported for theconventional PMCA (1.2 ag PrP^(Sc); Saa et al., (2006) J. Biol. Chem.281, 35245-35252), it can be achieved in two rPrP-PMCA rounds ofamplification over a total of about two days, whereas conventional PMCArequires seven rounds over a total of about 21 days (Saa et al., (2006)J. Biol. Chem. 281, 35245-35252). A single 50-hour round of conventionalPMCA takes about the same time as two rounds of rPrP-PMCA, but has a32,000-fold higher detection limit (1.6 pg; Saa et al., (2006) J. Biol.Chem. 281, 35245-35252). Without being bound by theory, it is believedthat the more rapid rPrP-PMCA reaction is facilitated in-part by thehigher concentration of rPrP-sen relative to that of PrP-sen in brainhomogenates.

It has also been found that the rPrP-PMCA/QUIC assay can performcross-species amplification of target PrP-res. In fact, rHaPrP-PMCA/QUICprovides a particularly suitable form of rPrP-res that promotesformation of PrP aggregates when incubated with a sample that containsPrP-res. rHaPrP appears to have a structure that promotes the formationof these aggregates with minimal formation of rPrP-res^((spon))byproduct. Hence rHaPrP can be used to amplify target PrP in a sampletaken from a species other than a hamster, such as a sample taken from ahuman, sheep, cow or cervid.

Another advantage of the rPrP-PMCA and QUIC assays is the ability todiscriminate between scrapie-infected and uninfected hamsters using 2-μlCSF samples (see FIG. 4). Because CSF is more accessible in liveindividuals than is brain tissue, it is an attractive biopsy specimenfor rPrP-PMCA- and QUIC-based diagnostic tests.

D. Recombinant Prion Protein

As described herein, the PrP-sen in used in rPrP-res PMCA reaction isrecombinant prion protein, for example prion protein from cellsengineered to over express the protein. Any prion protein sequence canbe used to generate the rPrP-sen, for instance: Xenopus laevis (GenbankAccession No: NP001082180), Bos taurus (Genbank Accession No: CAA39368),Danio verio (Genbank Accession No: NP991149), Tragelaphus strepsiceros(Genbank Accession No: CAA52781), Ovis aries (Genbank Accession No:CAA04236), Trachemys scripta (Genbank Accession No: CAB81568), Gallusgallus (Genbank Accession No: AAC28970), Rattus norvegicus NP036763),Mus musculus (Genbank Accession No: NP035300), Monodelphis domestica(Genbank Accession No: NP001035117), Homo sapiens (Genbank Accession No:BAA00011), Giraffa camelopardalis (Genbank Accession No: AAD13290),Oryctolagus cuniculus (Genbank Accession No: NP001075490), Macacamulatta (Genbank Accession No: NP001040617), Bubalus bubalus (GenbankAccession No: AAV30514), Tragelaphus imberbis (Genbank Accession No:AAV30511), Boselaphus tragocamelus (Genbank Accession No: AAV30507), Bosgarus (Genbank Accession No: AAV 30505), Bison bison (Genbank AccessionNo: AAV30503), Bos javanicus (Genbank Accession No: AAV30498), Synceruscaffer caffer (Genbank Accession No: AAV30492), Syncerus caffer nanus(Genbank Accession No: AAV30491), and Bos indicus (Genbank Accession No:AAV30489). In some embodiments, only a partial prion protein sequence isexpressed as rPrP-sen. For instance, in certain examples rPrP-senincludes amino acids 23-231 (SEQ ID NOS: 1, 2) of the hamster (SEQ IDNO: 8) or mouse (SEQ ID NO: 9) prion protein sequences, or thecorresponding amino acids of other prion protein sequences, for instanceamino acids 23-231 (SEQ ID NO: 3) of human (129M) prion protein (SEQ IDNO: 10), amino acids 23-231 (SEQ ID NO: 4) of human (129V) prion protein(SEQ ID NO: 11), amino acids 25-241 (SEQ ID NO: 5) of bovine(6-octarepeat) prion protein, amino acids 25-233 (SEQ ID NO: 6) of ovine(136A 154R 171Q) prion protein, or amino acids 25-234 (SEQ ID NO: 7) ofdeer (96G 132M 138S) prion protein. In general, the partial prionprotein sequence expressed as rPrP-sen corresponds to the polypeptidesequences of the natural mature full-length PrP^(C) molecule, meaningthat the rPrP-sen polypeptide lacks both the amino-terminal signalsequence and carboxy-terminal glycophosphatidylinositol-anchorattachment sequence. In another example, amino acids 30-231, 40-231,50-231, 60-231, 70-231, 80-231, or 90-231 of any one of human, human129V, bovine, ovine, or deer are utilized in the assays describedherein. One of skill in the art can readily produces these polypeptidesusing the sequence information provided in SEQ ID NOs: 1-11, or usinginformation available in GENBANK® (as available on Jul. 20, 2007).

The rPrP-sen can be a chimeric rPrP-sen, wherein a portion of theprotein is from one species, and a portion of the protein is fromanother species, can also be utilized. In one example about 10 to about90%, such as about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70% about 80% or about 90% of the rPrP-sen is from onespecies, and, correspondingly, about 90%, about 80%, about 70%, about60%, about 50%, about 40%, about 30%, about 20% or about 10% is fromanother species. Chimeric proteins can include, for example, hamsterrPrP-sen and rPrP-sen from another species, such as human PrP-sen.

In some embodiments, host cells are transformed with a nucleic acidvector that expresses the rPrP-sen, for example human, cow, sheep orhamster rPrP-sen. These cells can be mammalian cells, bacterial cells,yeast cells, insect cells, whole organisms, such as transgenic mice, orother cells that can serve as source of the PrP-sen. In particularexamples the cell is a bacterial cell, such as an E. coli cell. Raw celllysates or purified rPrP-sen from rPrP-sen expressing cells can be usedas the source of the non-pathogenic protein.

In some embodiments the recombinant protein is fused with an additionalamino acid sequence. For example, over expressed protein can be taggedfor purification or to facilitate detection of the protein in a sample.Some possible fusion proteins that can be generated include histidinetags, Glutathione S-transferase (GST), Maltose binding protein (MBP),green fluorescent protein (GFP), and Flag and myc-tagged rPrP. Theseadditional sequences can be used to aid in purification and/or detectionof the recombinant protein, and in some cases are subsequently removedby protease cleavage. For example, coding sequence for a specificprotease cleavage site can be inserted between the PrP-sen codingsequence and the purification tag coding sequence. One example for sucha sequence is the cleavage site for thrombin. Thus, fusion proteins canbe cleaved with the protease to free the PrP-sen from the purificationtag.

Any of the wide variety of vectors known to those of skill in the artcan be used to over-express rPrP-sen. For example, plasmids or viralvectors can be used. These vectors can be introduced into cells by avariety of methods including, but not limited to, transfection (forinstance, by liposome, calcium phosphate, electroporation, particlebombardment, and the like), transformation, and viral transduction.

Recombinant PrP-sen also can include proteins that have amino sequencescontaining substitutions, insertions, deletions, and stop codons ascompared to wild type sequences. In certain embodiment, a proteasecleavage sequence is added to allow inactivation of protein after it isconverted into prion form. For example, cleavage sequences recognized byThrombin, Tobacco Etch Virus (Life Technologies, Gaithersburg, Md.) orFactor Xa (New England Biolabs, Beverley, Mass.) proteases can beinserted into the sequence. In some embodiments, inactivation of proteinafter it is converted into prion form is unnecessary because therPrP-res^((Sc)) resulting from the reaction has little or noinfectivity.

Changes also can be made in the pPrP-sen protein coding sequence, forexample in the coding sequence for mouse, human, bovine, sheep, goat,deer and/or elk prion protein (GENBANK® accession numbers NM_(—)011170,NM_(—)183079, AY335912, AY723289, AY723292, AF156185 and AY748455,respectively, all of which are incorporated herein by reference, Jul.20, 2007). For example, mutations can be made to match a variety ofmutations and polymorphisms known for various mammalian prion proteingenes (see, for instance, Table 1). Furthermore, chimeric PrP moleculescomprising sequences from two or more different natural PrP sequences(for instance from different host species or strains) can be expressedfrom vectors with recombinant PrP gene sequences, and such chimeras canbe used for rPrP-PMCA and QUIC detection of prion from various species.Cells expressing these altered prion protein genes can be used as asource of the rPrP-sen, and these cells can include cells thatendogenously express the mutant rPrP gene, or cells that have been madeto express a mutant rPrP protein by the introduction of an expressionvector. Use of a mutated rPrP-sen can be advantageous, because some ofthese proteins are more easily or specifically converted toprotease-resistant forms, and thus can further enhance the sensitivityof the method.

In certain embodiments, cysteine residues are placed at positions 94 and95 of the hamster prion protein sequence in order to be able toselectively label the rPrP at those sites using sulfhydryl-reactivelabels, such as pyrene and fluorescein linked to maleimide-basedfunctional groups. In certain embodiments, these tags do not interferewith conversion but allow much more rapid, fluorescence-based detectionof the rPrP-PMCA reaction product. In one example, pyrenes in adjacentmolecules of rPrP-res are held in close enough proximity to allow eximerformation, which shifts the fluorescence emission spectrum in a distinctand detectable manner. Free pyrenes released from, or on, unconvertedrPrP-sen molecules are unlikely to form eximer pairs. Thus, therPrP-PMCA reaction can be run in a multiwell plate, digested withproteinase K, and then eximer fluorescence can be measured to rapidlytest for the presence of rPrP-res^((Sc)). Sites 94 and 95 were chosenfor the labels because the PK-resistance in this region of PrP-resdistinguishes rPrP-res^((Sc)) from rPrP-res^((spon)), giving rise to the17 kDa rPrP-res band. Other positions in the PK-resistant region(s) thatdistinguish the 17-kDa rHaPrP-res^((Sc)) fragment from allrHaPrP-res^((spon)) fragments also can work for this purpose.

TABLE 1 Pathogenic human Human Ovine Bovine mutations polymorphismspolymorphisms polymorphisms 2 octarepeat insert Codon Codon 171 5 or 6octarepeats 129 Met/Val Arg/Glu 4-9 octarepeat insert Codon 219 Codon136 Glu/Lys Ala/Val Codon 102 Pro-Leu Codon 105 Pro-Leu Codon 117Ala-Val Codon 145 Stop Codon 178 Asp-A Codon 180 Val-Ile Codon 198Phe-Ser Codon 200 Glu-Lys Codon 210 Val-Ile Codon 217 Asn-Arg Codon 232Met-Ala

E. Variant Prion Protein Sequences

As any molecular biology textbook teaches, a peptide of interest isencoded by its corresponding nucleic acid sequence (for instance, anmRNA or genomic DNA). Accordingly, nucleic acid sequences encoding prionproteins are contemplated herein, at least, to make and use the prionproteins of the disclosed compositions and methods.

In one example, in vitro nucleic acid amplification (such as polymerasechain reaction (PCR)) can be utilized as a method for producing nucleicacid sequences encoding prion proteins. PCR is a standard technique thatis described, for instance, in PCR Protocols: A Guide to Methods andApplications (Innis et al., San Diego, Calif.:Academic Press, 1990), orPCR Protocols, Second Edition (Methods in Molecular Biology, Vol. 22,ed. by Bartlett and Stirling, Humana Press, 2003).

A representative technique for producing a nucleic acid sequenceencoding a prion protein by PCR involves preparing a sample containing atarget nucleic acid molecule that includes the prion protein-encodingsequence. For example, DNA or RNA (such as mRNA or total RNA) can serveas a suitable target nucleic acid molecule for PCR reactions.Optionally, the target nucleic acid molecule can be extracted from cellsby any one of a variety of methods well known to those of ordinary skillin the art (for instance, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989;Ausubel et al., Current Protocols in Molecular Biology, Greene Publ.Assoc. and Wiley-Intersciences, 1992). Prion proteins are expressed in avariety of mammalian cells. In examples where RNA is the initial target,the RNA is reverse transcribed (using one of a myriad of reversetranscriptases commonly known in the art) to produce a double-strandedtemplate molecule for subsequent amplification. This particular methodis known as reverse transcriptase (RT)-PCR. Representative methods andconditions for RT-PCR are described, for example, in Kawasaki et al. (InPCR Protocols, A Guide to Methods and Applications, Innis et al. (eds.),21-27, Academic Press, Inc., San Diego, Calif., 1990).

The selection of amplification primers will be made according to theportion(s) of the target nucleic acid molecule that is to be amplified.In various embodiments, primers (typically, at least 10 consecutivenucleotides of prion-encoding nucleic acid sequence) can be chosen toamplify all or part of a prion-encoding sequence. Variations inamplification conditions may be required to accommodate primers andamplicons of differing lengths and composition; such considerations arewell known in the art and are discussed for instance in Innis et al.(PCR Protocols, A Guide to Methods and Applications, San Diego,Calif.:Academic Press, 1990). From a provided prion protein-encodingnucleic acid sequence, one skilled in the art can easily design manydifferent primers that can successfully amplify all or part of a prionprotein-encoding sequence.

As described herein, a number of prion protein-encoding nucleic acidsequences are known. Though particular nucleic acid sequences aredisclosed, one of skill in the art will appreciate that also providedare many related sequences with the functions described herein, forinstance, nucleic acid molecules encoding conservative variants of aprion protein. One indication that two nucleic acid molecules areclosely related (for instance, are variants of one another) is sequenceidentity, a measure of similarity between two nucleic acid sequences orbetween two amino acid sequences expressed in terms of the level ofsequence identity shared between the sequences. Sequence identity istypically expressed in terms of percentage identity; the higher thepercentage, the more similar the two sequences.

Methods for aligning sequences for comparison are well known in the art.Various programs and alignment algorithms are described in: Smith andWaterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol.Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins andSharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research16:10881-10890, 1988; Huang, et al., Computer Applications in theBiosciences 8:155-165, 1992; Pearson et al., Methods in MolecularBiology 24:307-331, 1994; Tatiana et al., (1999), FEMS Microbiol. Lett.,174:247-250, 1999. Altschul et al. present a detailed consideration ofsequence-alignment methods and homology calculations (J. Mol. Biol.215:403-410, 1990).

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST™, Altschul et al., J. Mol. Biol.215:403-410, 1990) is available from several sources, including theNational Center for Biotechnology Information (NCBI, Bethesda, Md.) andon the Internet, for use in connection with the sequence-analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe internet under the help section for BLAST™.

For comparisons of amino acid sequences of greater than about 30 aminoacids, the “Blast 2 sequences” function of the BLAST™ (Blastp) programis employed using the default BLOSUM62 matrix set to default parameters(cost to open a gap [default=5]; cost to extend a gap [default=2];penalty for a mismatch [default=−3]; reward for a match [default=1];expectation value (E) [default=10.0]; word size [default=3]; number ofone-line descriptions (V) [default=100]; number of alignments to show(B) [default=100]). When aligning short peptides (fewer than around 30amino acids), the alignment should be performed using the Blast 2sequences function, employing the PAM30 matrix set to default parameters(open gap 9, extension gap 1 penalties). Proteins with even greatersimilarity to the reference sequences will show increasing percentageidentities when assessed by this method, such as at least 50%, at least60%, at least 70%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% sequence identity to the sequence ofinterest.

For comparisons of nucleic acid sequences, the “Blast 2 sequences”function of the BLAST™ (Blastn) program is employed using the defaultBLOSUM62 matrix set to default parameters (cost to open a gap[default=11]; cost to extend a gap [default=1]; expectation value (E)[default=10.0]; word size [default=11]; number of one-line descriptions(V) [default=100]; number of alignments to show (B) [default=100]).Nucleic acid sequences with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 60%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, r at least 98%, or atleast 99% sequence identity to the prion sequence of interest.

Another indication of sequence identity is nucleic acid hybridization.In certain embodiments, prion protein-encoding nucleic acid variantshybridize to a disclosed (or otherwise known) prion protein-encodingnucleic acid sequence, for example, under low stringency, highstringency, or very high stringency conditions. Hybridization conditionsresulting in particular degrees of stringency will vary depending uponthe nature of the hybridization method of choice and the composition andlength of the hybridizing nucleic acid sequences. Generally, thetemperature of hybridization and the ionic strength (especially the Na⁺concentration) of the hybridization buffer will determine the stringencyof hybridization, although wash times also influence stringency.Calculations regarding hybridization conditions required for attainingparticular degrees of stringency are discussed by Sambrook et al. (ed.),Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, chapters 9 and11.

The following are representative hybridization conditions and are notmeant to be limiting.

Very High Stringency (detects sequences that share at least 90% sequenceidentity) Hybridization: 5x SSC at 65° C. for 16 hours Wash twice: 2xSSC at room temperature (RT) for 15 minutes each Wash twice: 0.5x SSC at65° C. for 20 minutes each High Stringency (detects sequences that shareat least 80% sequence identity) Hybridization: 5x-6x SSC at 65° C.-70°C. for 16-20 hours Wash twice: 2x SSC at RT for 5-20 minutes each Washtwice: 1x SSC at 55° C.-70° C. for 30 minutes each Low Stringency(detects sequences that share at least 50% sequence identity)Hybridization: 6x SSC at RT to 55° C. for 16-20 hours Wash at leasttwice: 2x-3x SSC at RT to 55° C. for 20-30 minutes each.

F. Prion Proteins

This disclosure further provides compositions and methods involving wildtype and recombinant prion proteins. In some embodiments, prion proteinvariants include the substitution of one or several amino acids foramino acids having similar biochemical properties (so-calledconservative substitutions). Conservative amino acid substitutions arelikely to have minimal impact on the activity of the resultant protein,such as it's ability to convert PrP-sen to PrP-res. Further informationabout conservative substitutions can be found, for instance, in BenBassat et al. (J. Bacteriol., 169:751-757, 1987), O'Regan et al. (Gene,77:237-251, 1989), Sahin-Toth et al. (Protein Sci., 3:240-247, 1994),Hochuli et al. (Bio/Technology, 6:1321-1325, 1988) and in widely usedtextbooks of genetics and molecular biology. In some examples, prionprotein variants can have no more than 3, 5, 10, 15, 20, 25, 30, 40, or50 conservative amino acid changes. The following table shows exemplaryconservative amino acid substitutions that can be made to a prionprotein, for instance the recombinant prion proteins shown in SEQ IDNOs: 1-7.

TABLE 2 Original Residue Conservative Substitutions Ala Ser Arg Lys AsnGln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn; Gln Ile Leu;Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr SerThr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

G. Purification of Recombinant Prion Protein

To purify PrP-sen from recombinant (or natural) sources, the compositionis subjected to fractionation to remove various other components fromthe composition. Various techniques suitable for use in proteinpurification are well known. These include, for example, precipitationwith ammonium sulfate, PTA, PEG, antibodies and the like, or by heatdenaturation followed by centrifugation; chromatography steps such asmetal chelate, ion exchange, gel filtration, reverse phase,hydroxylapatite, lectin affinity, and other affinity chromatographysteps; isoelectric focusing; gel electrophoresis; and combinations ofsuch and other techniques.

H. Sources of Samples for rPrP-Res Amplification Assays, Such asrPrP-PMCA and QUIC Assays

The samples analyzed using the methods described herein can include anycomposition capable of being contaminated with a prion. Suchcompositions can include tissue samples or bodily fluids including, butnot limited to, blood, lymph nodes, brain, spinal cord, tonsils, spleen,skin, muscles, appendix, olfactory epithelium, cerebral spinal fluid,urine, feces, milk, intestines, tears and/or saliva. Other compositionsfrom which samples can be taken for analysis, for instance, include foodstuffs, drinking water, forensic evidence, surgical implements, and/ormechanical devices.

I. Methods for Detecting rPrP-res^((Sc)) in rPrP-res AmplificationMixes, Such as rPrP-PMCA and QUIC Reaction Mixes

Once rPrP-res^((Sc)) has been generated using rPrP-res amplification,such as using rPrP-PMCA (such as the QUIC assay), rPrP-res^((Sc)) can bedetected in the reaction mix. Direct and indirect methods can be usedfor detection of rPrP-res^((Sc)) in a reaction mix or serial reactionmix. For methods in which rPrP-res^((Sc)) is directly detected,separation of newly-formed rPrP-res^((Sc)) from remaining rPrP-senusually is required. This typically is accomplished based on thedifferent natures of rPrP-res^((Sc)) versus rPrP-sen. For instance,rPrP-res^((Sc)) typically is highly insoluble and resistant to proteasetreatment. Therefore, in the case of rPrP-res^((Sc)) and rPrP-sen,separation can be by, for instance, protease treatment.

When rPrP-res^((Sc)) and rPrP-sen are separated by protease treatment,reaction mixtures are incubated with, for example, Proteinase K (PK). Anexemplary protease treatment includes digestion of the protein, forinstance, rPrP-sen, in the reaction mixture with 1-20 μg/ml of PK forabout 1 hour at 37° C. Reactions with PK can be stopped prior toassessment of prion levels by addition of PMSF or electrophoresis samplebuffer. Depending on the nature of the sample, incubation at 37° C. with1-50 μg/ml of PK generally is sufficient to remove rPrP-sen.

rPrP-res^((Sc)) also can be separated from the rPrP-sen by the use ofligands that specifically bind and precipitate the misfolded form of theprotein, including conformational antibodies, certain nucleic acids,plasminogen, PTA and/or various peptide fragments.

1. Western Blot

In some examples, reaction mixtures fractioned or treated with proteaseto remove rPrP-sen are then subjected to Western blot for detection ofrPrP-res^((Sc)) and the discrimination of rHaPrP-res^((Sc)) fromrHaPrP-res^((spon)). Typical Western blot procedures begin withfractionating proteins by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) under reducing conditions. The proteins arethen electroblotted onto a membrane, such as nitrocellulose or PVDF andprobed, under conditions effective to allow immune complex(antigen/antibody) formation, with an anti-prion protein antibody.Exemplary antibodies for detection of prion protein include the 3F4monoclonal antibody, monoclonal antibody D13 (directed against residues96-106 (Peretz et al. (2001) Nature 412, 739-743)), polyclonalantibodies R18 (directed against residues 142-154), and R20 (directedagainst C-terminal residues 218-232) (Caughey et al. (1991) J. Virol.65, 6597-6603).

Following complex formation, the membrane is washed to removenon-complexed material. An exemplary washing procedure includes washingwith a solution such as PBS/Tween, or borate buffer. The immunoreactivebands are visualized by a variety of assays known to those in the art.For example, the enhanced chemoluminesence assay (Amersham, Piscataway,N.J.) can be used.

If desired, prion protein concentration can be estimated by Western blotfollowed by densitometric analysis, and comparison to Western blots ofsamples for which the concentration of prion protein is known. Forexample, this can be accomplished by scanning data into a computerfollowed by analysis with quantitation software. To obtain a reliableand robust quantification, several different dilutions of the samplegenerally are analyzed in the same gel.

2. ELISA, Immunochromatographic Strip Assay, and Conformation DependentImmunoassay

As described above, immunoassays in their most simple and direct senseare binding assays. Specific non-limiting immunoassays of use includevarious types of enzyme linked immunosorbent assays (ELISAs),immunochromatographic strip assays, radioimmunoassays (RIA), andspecifically conformation-dependent immunoassays.

In one exemplary ELISA, anti-PrP antibodies are immobilized onto aselected surface exhibiting protein affinity, such as a well in apolystyrene microtiter plate. Then, a reaction mixture suspected ofcontaining prion protein antigen is added to the wells. After bindingand washing to remove non-specifically bound immune complexes, the boundprion protein can be detected. Detection generally is achieved by theaddition of another anti-PrP antibody that is linked to a detectablelabel. This type of ELISA is a simple “sandwich ELISA.” Detection alsocan be achieved by the addition of a second anti-PrP antibody, followedby the addition of a third antibody that has binding affinity for thesecond antibody, with the third antibody being linked to a detectablelabel.

In another exemplary ELISA, the reaction mixture suspected of containingthe prion protein antigen is immobilized onto the well surface and thencontacted with the anti-PrP antibodies. After binding and washing toremove non-specifically bound immune complexes, the bound anti-prionantibodies are detected. Where the initial anti-PrP antibodies arelinked to a detectable label, the immune complexes can be detecteddirectly. Again, the immune complexes can be detected using a secondantibody that has binding affinity for the first anti-PrP antibody, withthe second antibody being linked to a detectable label.

Another ELISA in which protein of the reaction mix is immobilizedinvolves the use of antibody competition in the detection. In thisELISA, labeled antibodies against prion protein are added to the wells,allowed to bind, and detected by means of their label. The amount ofprion protein antigen in a given reaction mix is then determined bymixing it with the labeled antibodies against prion before or duringincubation with coated wells. The presence of prion protein in thesample acts to reduce the amount of antibody against prion available forbinding to the well and thus reduces the ultimate signal. Thus, theamount of prion in the sample can be quantified.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating or binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one generallyincubates the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate are then washed to remove incompletely adsorbed material.Any remaining available surfaces of the wells are then “coated” with anonspecific protein that is antigenically neutral with regard to thetest antibodies. These include bovine serum albumin, casein, andsolutions of milk powder. The coating allows for blocking of nonspecificadsorption sites on the immobilizing surface, and thus reduces thebackground caused by nonspecific binding of antibodies onto the surface.

It is customary to use a secondary or tertiary detection means ratherthan a direct procedure with ELISAs, though this is not always the case.Thus, after binding of a protein or antibody to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with thebiological sample to be tested under conditions effective to allowimmune complex (antigen/antibody) formation. Detection of the immunecomplex then requires a labeled secondary binding ligand or antibody, ora secondary binding ligand or antibody in conjunction with a labeledtertiary antibody or third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and antibodies with solutions such as BSA, bovine gammaglobulin, milk proteins, and phosphate buffered saline (PBS)/Tween.These added agents also tend to assist in the reduction of nonspecificbackground. “Suitable” conditions also mean that the incubation is at atemperature and for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours, attemperatures preferably on the order of 25° C. to 27° C., or can beovernight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. An exemplary washingprocedure includes washing with a solution such as PBS/Tween or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes can bedetermined.

To provide a detecting means, the second or third antibody generallywill have an associated label to allow detection. In some examples, thisis an enzyme that will generate color development upon incubating withan appropriate chromogenic substrate. Thus, for example, the first orsecond immune complex is contacted and incubated with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (for instance, incubation for two hoursat room temperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, forinstance, by incubation with a chromogenic substrate such as urea andbromocresol purple or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonicacid) and H₂O₂, in the case of peroxidase as the enzyme label.Quantification is then achieved by measuring the degree of colorgeneration, for instance, using a visible spectra spectrophotometer.

J. rPrP-sen Labeling

In certain embodiments, the recombinant PrP-sen substrate protein can belabeled to enable high sensitivity of detection of protein that isconverted into rPrP-res^((Sc)). For example, rPrP-sen can beradioactively labeled, epitope tagged, or fluorescently labeled. Thelabel can be detected directly or indirectly. Radioactive labelsinclude, but are not limited to ¹²⁵I, ³²P, ³³P, and ³⁵S.

The mixture containing the labeled protein is subjected to an rPrP-Resamplification assay, such as rPrP-PMCA or QUIC, and the product detectedwith high sensitivity by following conversion of the labeled proteinafter removal of the unconverted protein for example by proteolysis.Alternatively, the protein can be labeled in such a way that a signalcan be detected upon the conformational changes induced duringconversion. An example of this is the use of FRET technology, in whichthe protein is labeled by two appropriate fluorophores, which uponrefolding become close enough to exchange fluorescence energy (see forexample U.S. Pat. No. 6,855,503).

In certain embodiments, cysteine residues are placed at positions 94 and95 of the hamster prion protein sequence in order to be able toselectively label the rPrP-sen at those sites using sulfhydryl-reactivelabels, such as pyrene and fluorescein linked to maleimide-basedfunctional groups. In certain embodiments, these tags do not interferewith conversion but allow much more rapid, fluorescence-based detectionof the rPrP-PMCA reaction product. In one example, pyrenes in adjacentmolecules of rPrP-res are held in close enough proximity to allow eximerformation, which shifts the fluorescence emission spectrum in a distinctand detectable manner. Free pyrenes released from, or on, unconvertedrPrP-sen molecules are unlikely to form eximer pairs. Thus, the rPrP-Resamplification reaction can be run in a multiwell plate, digested withproteinase K, and then eximer fluorescence can be measured to rapidlytest for the presence of rPrP-res^((Sc)). Sites 94 and 95 are chosen forthe labels because the PK-resistance in this region of PrP-resdistinguishes rPrP-res^((Sc)) from rPrP-res^((spon)), giving rise to the17 kDa rPrP-res band. Other positions in the PK-resistant region(s) thatdistinguish the 17-kDa rHaPrP-res^((Sc)) fragment from allrHaPrP-res^((spon)) fragments also can work for this purpose.

In certain other embodiments, the use of a fluorescently-tagged rPrP-sensubstrate for the reaction is combined with the use animmunochromatographic strip test with an immobilized rPrP-res specificantibody (for example, from Prionics AG, Schlieren-Zurich, Switzerland).Binding of the rPrP-res to the antibody is then detected with afluorescence detector.

K. Antibody Generation

In certain embodiments, the present disclosure involves antibodies, suchas antibodies that recognize PrP proteins. For example, antibodies areused in many of the methods for detecting prions (for instance, Westernblot and ELISA). In addition to antibodies generated against full lengthproteins, antibodies also can be generated in response to smallerconstructs comprising epitopic core regions, including wild-type andmutant epitopes.

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally,IgG and/or IgM are used because they are the most common antibodies inthe physiological situation and because they are most easily made in alaboratory setting. Monoclonal antibodies are recognized to have certainadvantages, for instance reproducibility and large-scale production. Themonoclonal antibodies can be of human, murine, monkey, rat, hamster,rabbit and even chicken origin.

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)2, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known. Means forpreparing and characterizing antibodies are also well known.

mAbs can be prepared through use of well-known techniques, such as thoseexemplified in U.S. Pat. No. 4,196,265. mAbs can be further purified, ifdesired, using filtration, centrifugation and various chromatographicmethods such as HPLC or affinity chromatography. Fragments of themonoclonal antibodies of the disclosure can be obtained from themonoclonal antibodies so produced by methods which include digestionwith enzymes, such as pepsin or papain, and/or by cleavage of disulfidebonds by chemical reduction. Alternatively, monoclonal antibodyfragments encompassed by the present disclosure can be synthesized usingan automated peptide synthesizer.

It also is contemplated that a molecular cloning approach can be used togenerate mAbs.

L. Screening for Modulators of Prion Function

The disclosed assay also can be used to identify compounds that modifythe ability of prions to replicate, such as compounds that would becandidates for the treatment of prion diseases. Thus, the method forscreening compounds includes performing an rPrP-Res amplification assayon control reaction mixtures, and reaction mixtures including the testcompound are accessed for levels of rPrP-res^((Sc)) followingamplification. When a difference between the levels of rPrP-res^((Sc))in the test versus control reaction mixtures is detected, compoundscould be identified that either enhance or inhibit conversion ofrPrP-sen to rPrP-res^((Sc)). These assays can include random screeningof large libraries of candidate substances; alternatively, the assayscan be used to focus on particular classes of compounds selected with aneye towards structural attributes that are believed to make them morelikely to modulate the function of prions.

By function, it is meant that one can determine the efficiency ofconversion by assaying conversion of a standard amount of rPrP-sen intorPrP-res^((Sc)) by a known amount of prion. This can be determined by,for instance, quantitating the amount of rPrP-res^((Sc)) in a reactionmix following a certain number of cycles of rPrP-PMCA or QUIC.

As used herein, the term “candidate substance” refers to any moleculethat potentially can inhibit or enhance prion function activity. Thecandidate substance can be a protein or fragment thereof, a smallmolecule, a polymer or even a nucleic acid molecule. The most usefulpharmacological compounds can be compounds that are structurally relatedto prion protein or prion protein ligands. Using lead compounds to helpdevelop improved compounds is known as “rational drug design,” andincludes not only comparisons with known inhibitors and activators, butpredictions relating to the structure of target molecules. The goal ofrational drug design is to produce structural analogs of biologicallyactive polypeptides or target compounds. By creating such analogs, it ispossible to fashion drugs that are more active or stable than thenatural molecules, and that have different susceptibility to alterationor which can affect the function of various other molecules. In oneapproach, one generates a three-dimensional structure for a targetmolecule, or a fragment thereof, for instance by x-ray crystallography,computer modeling, or by a combination of both approaches.

It also is possible to use antibodies to ascertain the structure of atarget compound activator or inhibitor. In principle, this approachyields a pharmacore upon which subsequent drug design can be based. Itis possible to bypass protein crystallography altogether by generatinganti-idiotypic antibodies to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site of ananti-idiotype would be expected to be an analog of the original antigen.The anti-idiotype could then be used to identify and isolate peptidesfrom banks of chemically- or biologically-produced peptides. Selectedpeptides would then serve as the pharmacore. Anti-idiotypes can begenerated using the methods described herein for producing antibodies,using an antibody as the antigen.

Alternatively, small molecule libraries can be acquired that arebelieved to meet the basic criteria for useful drugs in an effort toidentify useful compounds by large-scale screening. Screening of suchlibraries, including combinatorially generated libraries (for instance,peptide libraries), is a rapid and efficient way to screen large numberof related (and unrelated) compounds for activity. Combinatorialapproaches also lend themselves to rapid evolution of potential drugs bythe creation of second, third and fourth generation compounds modeled onactive, but otherwise undesirable compounds.

Candidate compounds can include fragments or parts ofnaturally-occurring compounds, or can be found as active combinations ofknown compounds, which are otherwise inactive. Compounds isolated fromnatural sources, such as animals, bacteria, fungi, plant sources,including leaves and bark, and marine samples, can be assayed ascandidates for the presence of potentially useful pharmaceutical agents.It will be understood that the pharmaceutical agents to be screened alsocould be derived or synthesized from chemical compositions or man-madecompounds. Thus, it is understood that the candidate substanceidentified by the present disclosure can be peptide, polypeptide,polynucleotide, glycans, synthetic polymers, small molecule inhibitorsor any other compound(s) that can be designed through rational drugdesign starting from known inhibitors or stimulators. Other suitablemodulators include antibodies (including single chain antibodies), eachof which would be specific for the target molecule. Such compounds aredescribed in greater detail above.

In addition to the modulating compounds initially identified, othersterically similar compounds can be formulated to mimic the key portionsof the structure of the modulators. Such compounds, which can includepeptidomimetics of peptide modulators, can be used in the same manner asthe initial modulators. Preferred modulators of prion replication wouldhave the ability to cross the blood-brain barrier since a large numberof prion manifest themselves in the central nervous system.

An inhibitor can be one that exerts its activity directly on thePrP-res, on the PrP-sen, or on factors required for the conversion ofPrP-sen to PrP-res. Regardless of the type of inhibitor or activatoridentified by the present screening methods, the effect of theinhibition or activation by such a compound results in altered prionamplification or replication as compared to that observed in the absenceof the added candidate substance.

M. Kits

Any of the compositions described herein can be included in a kit forcarrying out rPrP-PCMA or QUIC. In a non-limiting example, recombinantPrP-sen, prion conversion factors, decontamination solution, and/orconversion buffer with a metal chelator are provided in a kit. The kitfurther can include reagents for expressing or purifying rPrP-sen. Thekit also can include pre-labeled rPrP-sen or reagents that can be usedto label the rPrP-sen, with for example, radio isotopes or fluorophores.

In some embodiments, kits are provided for amplification and detectionof prion in a sample. In these embodiments, a kit can include, insuitable container, one or more of the following: 1) a conversionbuffer; 2) decontamination solution; 3) a positive control, prioncontaining sample; 4) a negative control sample, not containing prion;or 5) reagents for detection of rPrP-res.

Regents for the detection of prions can include one or more of thefollowing: pre coated microtiter plates for ELISA and/or CDI detectionof rPrP-res; or antibodies for use in ELSA, CDI, stripimmunochromatography or Western blot detection methods.

Additionally, kits of the disclosure can contain one or more of thefollowing: protease free water; copper salts for inhibiting rPrP-resamplification; EDTA solutions for enhancing prion replication;Proteinase K for the separation of rPrP-res from rPrP-sen; fractionationbuffers for the separation of rPrP-res from rPrP-sen, modified, orlabeled proteins (to increase sensitivity of detection); or conversionfactors (to enhance efficiency of amplification).

In certain embodiments, the conversion buffer is supplied in a “readyfor amplification format” where it is allocated in a microtiter platesuch that the sample and rPrP-sen can be added to first well, andsubjected to primary reaction and amplification. Thereafter a portion ofthe reaction mix is moved to an adjacent well with additional rPrP-senadded for serial rPrP-res amplification. These steps can be repeatedacross the microtiter plate for multiple serial amplifications.

The components of the kits can be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, plate, flask, bottle, syringe or othercontainer, into which a component can be placed, and optionally,suitably aliquoted. Where there is more than one component in the kit(labeling reagent and label can be packaged together), the kit alsogenerally will contain a second, third or other additional containerinto which the additional components can be separately placed. However,various combinations of components can be included in a vial. The kitsalso typically will include a means for containing proteins, and anyother reagent containers in close confinement for commercial sale. Suchcontainers can include injection or blow-molded plastic containers intowhich the desired vials are retained.

When components of the kit are provided in one and/or more liquidsolutions, the liquid solution is typically an aqueous solution that issterile and proteinase free. In some cases protein-based compositionsare lyophilized to prevent degradation and/or the kit or componentsthereof can be stored at a low temperature (for instance, less thanabout 4° C.). When reagents and/or components are provided as a drypowder and/or tablets, the powder can be reconstituted by the additionof a suitable solvent. It is envisioned that the solvent also can beprovided in another container means.

N. rPrP-Res Amplification Assays (rPrP-PMCA/QUIC Assays) Using Samplesfrom Humans, Bovines, and Other Species

As described above, it is desirable to carry out rPrP-PMCA and/or QUICassays in a variety of species. For instance, the assays are useful inscreening bovine, sheep, and cervid individuals or populations for priondiseases, for example to ensure the safety of the food supply. In someinstances, rPrP-PMCA/QUIC assays are useful for diagnosing prion diseasein a human or veterinary subject.

The rPrP-sen chosen may be chosen from the same species as the testsample, or it may be of a different species. For example, a hamster ormouse rPrP-sen can be used to amplify a human or sheep test sample. Inparticular examples hamster rPrP (rHaPrP) is used because it isparticularly effective in amplification reactions, (such as QUIC) notonly of hamster PrP-res but of PrP-res from humans and sheep as well.For those embodiments in which the rPrP-sen is from the same species asthe test sample, it is also desirable to create rPrP-sen from a varietyof species that may be tested. As described above in greater detail, ingeneral, the partial prion protein sequence expressed as rPrP-sencorresponds to the polypeptide sequences of the natural maturefull-length PrP^(C) molecule, meaning that the rPrP-sen polypeptidelacks both the amino-terminal signal sequence and carboxy-terminalglycophosphatidylinositol-anchor attachment sequence. Thus, in someembodiments, a hamster rPrP includes amino acids 23-231 (SEQ ID NO: 1)of hamster prion protein sequence (SEQ ID NO: 8), a bovine rPrP-senincludes amino acids 25-241 (SEQ ID NO: 5) of a bovine prion proteinsequence, whereas a human rPrP-sen includes amino acids 23-231 (SEQ IDNOs: 3, 4) of a human prion protein sequence (SEQ ID NOs: 10, 11), anovine rPrP-sen includes amino acids 25-233 (SEQ ID NO: 6) of an ovineprion protein sequence, and a cervid rPrP-sen includes amino acidresidues 25-234 (SEQ ID NO: 7) of a cervid prion protein sequence orresidues 90-231 of the hamster sequence (SEQ ID NO: 8). However, therPrP protein is not limited to these particular portions of eachsequence. Other exemplary portions are disclosed above.

As described above in greater detail, the test sample can be any tissuesample from a human or veterinary subject, for instance a brain sample,peripheral organ sample (such as blood, tonsil, spleen, or anotherlymphoid organ), various excretia or a CSF sample. In the case of livingsubjects, blood, excretia, or CSF samples are easily obtained withrelatively non-invasive techniques. In the case of samples from deceasedsubjects, brain or other tissue samples are easily obtained.

Once the rPrP-sen and test samples have been obtained, an rPrP-PMCA orQUIC assay is performed as described in detail above. Results generallyare available within 24-72 hours, which greatly speeds diagnosis,treatment, and/or disease containment/decontamination efforts.

The following Examples are provided to illustrate certain particularfeatures and/or embodiments. These Examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 Materials and Methods

This example describes materials and methods used to carry out Examples2-8. Although particular methods are described, it is understood thatother methods can be used.

Recombinant Prion Protein Expression and Purification

DNA sequences coding for hamster (GENBANK® Accession No. M14054) andmouse (GENBANK® Accession No. BC006703) prion protein residues 23-230 or90-230 were amplified by standard PCR, ligated into the Kanamycinselective pET41 vector (EMD Biosciences) as NdeI/HindIII inserts, andtheir sequences were verified. After transforming the plasmids into E.coli Rosetta cells (EMD Biosciences), the rPrP-sen was expressed usingthe Overnight Express Autoinduction system according to the instructionsfrom the manufacturer (EMD Biosciences). A typical mass of wet cellpaste was 8-9 grams per Liter of Luria-Bertani media. Cell pellets werelysed with BugBuster™ and Lysonase™ (EMD Bioscieces). Approximately 25mL of BugBuster™ with 50 μL of lysonase and one Complete EDTA-freeprotease inhibitor tablet (Roche) was used for each gram of harvestedbacterial cells. This lysis mixture was stirred with the bacterial cellsin an ice bath. This mixture was then subjected to periodic sonication(15 pulses of 15 seconds over 30 minutes at full power) to facilitatelysis. Inclusion bodies containing rPrP-sen were isolated bycentrifugation and then were twice washed with 0.1×BugBuster™ andpelleted by centrifugation in 50 mL centrifuge tubes. The enriched rPrPwas further purified by modifications to the method of Zahn et al.,(1997) FEBS Lett. 417, 400-404. The washed inclusion bodies were thensuspended in aqueous 8 M Guanidine hydrochloride and this mixture waspelleted by centrifugation to remove cell debris. The supernatantcontaining denatured rPrP-sen was stirred with Ni-NTA Superflow (Qiagen)resin and then loaded onto an XK16/20 column (GE Healthcare) and thenwashed with denaturing buffer (6 M Guanidine hydrochloride, 100 mMsodium phosphate, 10 mM Tris, pH 8.0) and refolded with a lineargradient over 6 hours at a flow rate of 0.75 to 1 mL/minute using anAKTA Explorer 10. The protein was then eluted with 100 mM sodiumphosphate (pH 5.8), 500 mM imidazole, 10 mM Tris. Pooled fractions werediluted to 0.2 mg/ml with water, filtered and dialyzed against 10 mMphosphate. The 10 mM phosphate dialysis buffer was diluted from a 1 Mstock of the same buffer (pH 5.8) immediately prior to dialysis. Afterdialysis against a total of 4 L (2×2 L) at 4° C., with the secondtreatment overnight, the protein solution was sterile filtered through a0.22 μm 150 mL filter unit (Millipore) and the protein concentration ofrPrP was determined by the method of Bradford or by A_(280nm). Purity ofthe final protein preparations was estimated at 99% when analyzed bySDS-PAGE, immunoblotting and MALDI mass spectrometry.

Differences of this method to that of Zahn et al. include the isolationof inclusion bodies that were not isolated in Zahn et al., lysis innon-denaturing Bug Buster instead of lysis in a denaturing buffer, theelimination of the use of a glutathione denaturing buffer, and theelimination of the histidine tag.

rPrP-PMCA

rPrP-PMCA reactions were prepared in 0.2 ml PCR tubes as 80 μl solutionscontaining PBS, pH 7.4, containing 0.05% (w/v) SDS and 0.05% TritonX-100(TX-100), except as shown in FIG. 1, where 0.1% of each detergent wasused. rHaPrP-sen was present at 0.1 mg/ml (4 μM). The reactions wereseeded with brain homogenate from Syrian golden hamsters affected withthe 263K scrapie strain (ScBH) or purified PrP^(Sc) (HaPrP^(Sc)) fromthe same source (Raymond & Chabry in Techniques in Prion Research (eds.Lehmann & Grassi) 16-26 (Birkhauser Verlag, Basel, 2004)). The PrP^(Sc)concentration in the ScBH was estimated by semiquantitativeimmunoblotting against purified HaPrP^(Sc) standards. Reactions wereimmersed in water at 37° C. and subjected to repeated cycles ofsonication (Misonix Model 3000), based on previous methods (see, forinstance, Saa et al., (2006) J. Biol. Chem. 281, 35245-35252) with minormodifications. In brief, sonication was performed over 24 hours(constituting one round) with 40 second pulses every 60 minutes atmaximum power. Unsonicated controls were incubated at 37° C.

Although rPrP-PMCA will work under a variety of conditions, the optimalconditions that supported specific PrP^(Sc)-seeded rPrP-PMCA include thecombination of about 0.05-0.1% of an anionic detergent such as SDS andabout 0.05-0.1% of a nonionic detergent such as TX-100. These conditionsare particularly effective at promoting the formation ofrHaPrP-res^((Sc)) (notably the 17 kDa PK-resistant species), whilereducing rHaPrP-res^((spon)) formation within the first 24 hours ofunseeded reactions. Previous studies showed that prion proteinaggregation can be prompted by low concentrations of anionic detergents(Xiong et al., (2001) J. Neurochem. 79, 669-678). Other conditions canpromote the spontaneous formation of rPrP-res that includes a 17-kDafragment (Bocharova et al. (2006) J. Biol. Chem. 281, 2373-2379), soseeding with PrP^(Sc) is not always required for the formation ofrPrP-res with a banding pattern like that of rHaPrP-res^((Sc)). However,under the rPrP-PMCA conditions set forth in this example, PrP^(Sc)seeding is required, allowing for clear and consistent discriminationbetween HaPrP^(Sc)-seeded and unseeded reactions. Without being bound bytheory, it is believed that these specific detergent conditions canpartially unfold rPrP-sen, allowing productive contacts between PrP^(Sc)and rPrP-sen that would not otherwise occur spontaneously betweenrPrP-sen molecules.

QUIC

A different method from PMCA uses Quaking Induced Conversion (QUIC), inwhich shaking of the reaction mixture replaces sonication fordisaggregating aggregates formed during cyclic amplification. Of courseboth shaking and sonication can be used in an amplification reaction,for example in alternating cycles. In the particular examples of QUICdisclosed herein only shaking of reaction vessels is used.

Either purified PrP^(Sc) or scrapie brain homogenate were used to seedthe conversion of rPrP-sen to protease-resistant forms in reactionsperformed in 0.1% sodium dodecyl sulfate and 0.1% Triton X-100, in PBSat 37° C. in 0.5 ml tubes. Tube shaking was done at 1500 rpm in anEppendorf Thermomixer R. Proteinase K digestions and immunoblotting wereperformed as described in the step-by-step protocol, below.

For comparing PK-resistant QUIC reaction products, 24-hour unshakenreactions and reactions were shaken with or without 0.1 mm glass celldisruption beads (Scientific Industries). These reactions were seededwith 10 ng of purified hamster PrP^(Sc) with 0.2 mg/ml hamster rPrP-senand a 50 μl reaction volume. The tubes were subjected to cycles of 2minutes of shaking and 28 minutes without shaking. C-terminal antibodyR20 was used for the immunoblot.

For 20-hour QUIC reactions performed with the varying rPrP-senconcentrations, reaction volumes, and seed amounts, the seed amountsapproximate the estimated quantity of PrP^(Sc) added in 2-μl aliquotsdilutions of scrapie brain homogenate (in 1% normal brain homogenate).The tubes were subjected to cycles of 10 seconds of shaking and 110seconds without shaking. R20 was used for the immunoblot.

For extended reactions to QUIC sensitivity to small amounts of scrapiebrain homogenate seed, 65-hour and 95-hour QUIC reactions were carriedout as described above, and 0.2 mg/ml rPrP-sen, were used for 100-μlreaction volumes. Scrapie brain homogenate seed dilutions containing thedesignated amount of PrP^(Sc), were subjected to cycles of 10 secondsshaking and 110 seconds without shaking.

In other examples, 48-hour reaction times were used with reduceddetergent concentrations (0.05% SDS and 0.05% Triton X-100). For thesecond round, 10% of the volume of the first round reaction productswere diluted into 9 volumes of reaction buffer containing freshrPrP-sen. PK-digestions and immunoblotting using either R20 or D13primary antibodies were performed as described below.

For seeding with CSF samples, aliquots (2 μl) of CSF taken from normalhamsters (n=3) or hamsters in the clinical phase of scrapie (n=6) wereused to seed QUIC reactions using the conditions, and immunoblots werecarried out using the PK-digested products of the first 48-hour round.Ten percent of each first round reaction volume was used to seed asecond 48-hour round of QUIC. Antibodies R20 and D13 were used for theimmunoblots.

CSF Collection

Hamsters were heavily sedated with isofluorane and exsanguinated usingcardiac puncture. Skin and muscles at the back of the neck weredissected away avoiding blood vessels and meninges. A small hole wasmade at the medial aperture in the meninges using a 26¾ G needle and aDrummond micropipette was quickly inserted into the hole. CSF filled themicropipette by capillary action. Rocky Mountain Laboratories is anAALAC-accredited facility, and all animal procedures were approved bythe institution's Animal Use and Care Committee.

Proteinase K Digestion, SDS-PAGE and Western Blotting

At the end of the reaction, 5 μl of the reaction sample (1 μg of rPrP)was diluted five-fold in PBS with 0.1% SDS and digested with thespecified PK:rHaPrP ratio (0.025:1=1 μg/ml of PK, 0.25:1=10 μg/ml of PK,or 0.5:1=20 μg/ml of PK) for 1 hour at 37° C. PEFABLOC®(4-(2-Aminoethyl)-benzensulfonyl fluoride (Roche) was then added to afinal concentration of 4 mM. For those samples analyzed by westernblotting, 20 μg of thyroglobulin was added and the protein wasprecipitated with 4 volumes of methanol and stored at −20° C. prior tocentrifugation and aspiration of the methanol. Pellets were suspended insample buffer (4 M urea, 4% SDS, 2% β-mercaptoethanol, 8% glycerol,0.02% bromophenol blue and 50 mM Tris-HCl pH6.8), subjected to SDS-PAGEusing 10% BisTris NUGPAGE® (polyacrylamide) gels (Invitrogen), andtransferred to IMMOBILON™ P membrane (Millipore). The membrane wasprobed with D13 (Peretz et al. (2001) Nature 412, 739-743), R20 (Caugheyet al., (1991) J. Virol. 65, 6597-6603), or R18 antibodies at 1:10,000dilutions as specified, and visualized by ATTOPHOS® AP FluorescentSubstrate System (2′-[2-benzothiazoyl]-6′-hydroxybenzothiazole phosphate[BBTP]) (Promega) according to the manufacturer's recommendations. Forsilver staining, methanol precipitation was omitted and the PK-digestedsamples were mixed with 5× sample buffer, boiled, and analyzed bySDS-PAGE.

Electron Microscopy

rHaPrP-res^((spon)) and rHaPrP-res^((Sc)) from fourth round reactionswere treated with PK (PK:PrP ratio of 0.025:1) at 37° C. for one hour,after which 5 mM PEFABLOC®_(4-(2-Aminoethyl)-benzensulfonyl fluoride)was added. These and PK untreated samples were pelleted bycentrifugation for 30 minutes at 16,100 g, washed twice with PBS orwater, and sonicated for one minute. The samples were then settled ontoFormvar-coated grids for 15 minutes, washed three times with sterilewater, and stained with methylamine tungstate for one minute. Excessstain was removed by filter paper and the samples were dried at roomtemperature. Images were obtained with an 80 kV in a Hitachi H-7500electron microscope and an AMT XR-100 digital camera system (AdvancedMicroscopy Techniques, Danvers, Mass.).

Spectral Analysis

rHaPrP-res^((spon)) and rHaPrP-res^((Sc)) (seeded with purified HaPrPSc)from third round reactions were pelleted by centrifugation for 30minutes at 16,100 g and twice washed in 10 μl of sterile water. Slurriedpellets were applied to a Golden Gate Single Reflection DiamondAttenuated Total Reflectance unit purged with dehydrated air and driedunder a stream of nitrogen. Data collection was performed using a System2000 IR instrument (Perkin-Elmer) with a liquid nitrogen cooled nbl MCTdetector and the following parameters: 20° C., 1 cm⁻¹ resolution, 5 cm/soptical path difference velocity, 500 scans 1800-1400 cm⁻¹ scan range,and 0.5 cm⁻¹ data interval. Primary spectra were obtained by subtractingthe corresponding buffer and water vapor spectra. Fourier-selfdeconvoluted spectra were calculated from the primary difference spectrausing a gamma of 19.5 and a smoothing length of 95%. The software usedfor spectral analyses was Spectrum v2.00 (Perkin-Elmer).

Example 2 Spontaneous Conversion of rPrP-Sen

This Example describes the identification of an exemplary set ofreaction conditions that allow clear discrimination betweenPrP^(Sc)-seeded and unseeded reaction products. Although particularreaction conditions are specified, one will recognize that otherreaction conditions can be used.

Development of a PMCA-like reaction for PrP^(Sc) amplification usingrPrP-sen as a substrate requires conditions that allow for cleardiscrimination between PrP^(Sc)-seeded and unseeded reaction products.Initial trials revealed that in 0.1% SDS with periodic sonications,bacterially expressed recombinant mouse PrP-sen (rMoPrP-sen; FIG. 5) andhamster PrP-sen (rHaPrP-sen) converted spontaneously to thioflavinT-positive, proteinase K (PK)-resistant forms designatedrMoPrP-res^((spon)) and rHaPrP-res^((spon)), respectively. The fragmentsgenerated by PK-digestion of rMoPrP-res^((spon)) and rHaPrP-res^((spon))were 10-12 kDa, that is, much smaller than the ˜17-19 kDa fragmenttypical of unglycosylated scrapie PrP^(Sc) and PrP^(Sc)-inducedrPrP-res⁸⁻¹⁰. When seeded into fresh solutions of rMoPrP-sen andrHaPrP-sen, respectively, rMoPrP-res^((spon)) and rHaPrP-res^((spon))elicited more thioflavin T-positive rPrP-res^((spon)), even withoutsonication (FIG. 6). However, the addition of 0.1% TX-100 to the 0.1%SDS permitted seeded rPrP-res^((spon)) accumulation, but often delayedits spontaneous formation for >24 hours even in sonicated reactions.Thus, these conditions were selected for subsequent attempts to seedrHaPrP-sen conversion with PrP^(Sc).

Example 3 Seeding of rPrP-Sen Conversion by PrP^(Sc)

This example demonstrates that scrapie PrP^(Sc) can seed the conversionof rPrP-sen to rPrP-res.

Scrapie PrP^(Sc) purified from hamster brains (HaPrP^(Sc); Raymond &Chabry in Techniques in Prion Research (eds. Lehmann & Grassi) 16-26(Birkhauser Verlag, Basel, 2004)) was used to seed the conversion ofrHaPrP-sen. PK-resistant fragments seeded by PrP^(Sc)(rHaPrP-res^((Sc)), where ^((Sc)) refers to seeding by PrP^((Sc)) weregenerated with seed-to-substrate ratios of 1:100 (400 ng HaPrP^(Sc)) and1:1,000 (40 ng HaPrP^(Sc)) in both the unsonicated and sonicatedreactions, but, when sonicated were much more abundant and lessdependent on the amount of seed (FIG. 1A). When analyzed byimmunoblotting using an anti-PrP antibody R20 directed toward C-terminalresidues 219-232, rHaPrP-res^((Sc)) consisted of 4 PK-resistantfragments (11, 12, 13 and 17 kDa). In contrast, and as expected, theunseeded reactions gave either no PK-resistant bands (FIG. 1A) or, morerarely, rHaPrP-res^((spon)) with only the smaller 10-, 11- and 12-kDafragments (FIG. 1A). The 17-kDa rHaPrP-res^((Sc)) band also was notobserved in the absence of rHaPrP-sen substrate, demonstrating that theHaPrP^(Sc) seed itself did not display this band (FIG. 1A).Collectively, these data demonstrate that HaPrP^(Sc)-seeded rPrP-senconversion reactions can be distinguished from unseeded reactions byimmunoblot comparison of the PK-resistant banding patterns. Most notablewas the formation of the 17 kDa-band in the HaPrP^(Sc)-seeded reactionsas has been observed previously in substoichiometric conversionreactions with rPrP-sen (Iniguez et al., (2000) J. Gen. Virol. 81,2565-2571; Kirby et al., (2003) J. Gen. Virol. 84, 1013-1020; Eiden etal., (2006) J. Gen. Virol. 87, 3753-3761).

The ability of rHaPrP-res^((Sc)) to seed additional rounds ofrHaPrP-res^((Sc)) amplification was tested by diluting products of thefirst-round HaPrP^(Sc)-seeded reaction seeded (FIG. 1A) into freshrHaPrP-sen substrate. For brevity, the term “rPrP-PMCA” is used whenreferring to the use of rPrP-sen as a substrate in combination withperiodic sonication and (optionally) cyclic dilutions of reactionproducts into fresh substrate to detect PrP^(Sc) and amplifyrHaPrP-res^((Sc)). Without sonication, the rHaPrP-res^((Sc)) produced inboth the first and second rounds decreased with dilution of the seed(FIGS. 1A, 1B). With sonication, the yield was less dependent upon seedconcentration, with similarly high levels of rHaPrP-res^((Sc)) producedat each dilution (FIGS. 1A, 1B). Similar levels of rHaPrP-res^((Sc))were produced in each of five consecutive rounds of amplification withthe products of each round diluted 1,000-fold into newly preparedrHaPrP-sen. Overall, periodic sonication reduced the amount ofHaPrP^(Sc) required to initiate robust rHaPrP-res^((Sc)) generation.

To further clarify the difference in the PK susceptibility betweenrHaPrP-res^((Sc)) and rHaPrP-res^((spon)), immunoblots were performedwith additional antibodies (FIG. 1C). Monoclonal antibody D13 (directedagainst residues 96-106 (Peretz et al. (2001) Nature 412, 739-743))specifically recognized the PrP^(Sc)-induced 17 kDa band but not thelower molecular weight fragments. In contrast, the polyclonal antibodyR18 (directed against residues 142-154 (Peretz et al. (2001) Nature 412,739-743)) recognized 17 kDa, 13 kDa and 12 kDa fragments inrHaPrP-res^((Sc)) and 12 kDa fragments in rHaPrP-res^((spon)). TheC-terminal antibody R20 reacted with all of the rHaPrP-res fragments,including the shortest 10 kDa fragment that appears to be specific forrHaPrP-res^((spon)), indicating these fragments differed primarily attheir N-termini. Distinct fragment patterns were observed forrHaPrP-res^((Sc)) and rHaPrP-res^((spon)) over a wide range of PK:rPrPratios (FIG. 1C) and detergent compositions (FIG. 7). In agreement withthe R20 immunoblots (FIG. 1C), silver-stained SDS-PAGE gels ofPK-digested third-round reaction products confirmed thatrHaPrP-res^((Sc)) comprised primarily the 11, 12, 13 and 17 kDa bandswhile rHaPrP-res^((spon)) comprised the 10, 11 and 12 kDa bands (FIG.1D). Thus, PrP^(Sc)-seeded and non-seeded reaction products differed intheir susceptibility to proteolytic cleavage, providing compellingevidence for fundamental differences in conformation.

Example 4 Ultrasensitive Detection of PrP^(Sc)

To determine the minimum amount of PrP^(Sc) detectable by rPrP-PMCA,scrapie brain homogenates (ScBH) were diluted serially with 1% normalbrain homogenate (NBH) and were used to seed rPrP-PMCA reactions. ThePK-treated products were analyzed by immunoblotting with D13 antibody.After a single round, the 17-kDa rHaPrP-res^((Sc)) band was detected inreactions seeded with a 6×10⁻⁸ dilution of ScBH containing ≧10 fg (10⁻¹⁵g) of PrP^(Sc) (FIG. 2A). With a second round of amplification seededwith 10% of the first round reaction products, the sensitivity improved,allowing consistent detection of dilutions of ScBH containing ˜50 ag(5×10⁻¹⁷ g), or 1,000 molecules, of the original HaPrP^(Sc) seed (FIG.2B). This amount of ScBH typically would contain an average of 0.003i.c. LD₅₀ (a dose lethal to 50% of inoculated hamsters) of scrapieinfectivity according to 3 independent end-point dilution bioassays ofother brain homogenates stocks prepared from Syrian hamsters in theclinical phase of scrapie (Silveira et al. (2005) Nature 437, 257-261).A subset of replicate reactions were positive with further dilutions ofScBH containing 10-20 ag (nominally) of HaPrP^(Sc). However, none of theNBH controls or samples seeded with more dilute ScBH gave detectable17-kDa bands. Further rounds of rPrP-PMCA did not increase thesensitivity of PrP^(Sc) detection. These results indicate that rPrP-PMCAcan detect sublethal amounts of scrapie-infected tissue.

Example 5 Electron Microscopy and Fourier Transform InfraredSpectroscopy (FTIR)

This Example describes electron microscopy and Fourier transforminfrared spectroscopy of rHaPrP-res^((Sc)) and rHaPrP-res^((spon)).

Negative-stained transmission electron microscopy of rHaPrP-res^((Sc))and rHaPrP-res^((spon)) revealed that both contained short bundles offibrillar aggregates, which were especially apparent after PK treatments(FIG. 8). However, other than a tendency of rHaPrP-res^((Sc)) to bebundled laterally more than rHaPrP-res^((spon)), we observed noconsistent ultrastructural differences between the two types of fibrils.

Comparisons of the secondary structures of rHaPrP-res^((Sc)) andrHaPrP-res^((spon)) by FTIR provided additional evidence that theydiffer in conformation (FIG. 9). The value of rHaPrP-res^((Sc)) as aPrP^(Sc) surrogate will depend in part upon the extent to which itmimics PrP^(Sc) conformationally. Comparison of rHaPrP-res^((Sc)) versusrHaPrP-res^((spon)) showed that the former has a distinct PK-resistantfragmentation pattern and an FTIR band at 1637 cm⁻¹ (FIG. 9) that isreminiscent of 263K HaPrP^(Sc) itself^(23,24). There are alsodifferences between the rPrP-res fragment pattern and FTIR spectra ofrHaPrP-res^((Sc)) and HaPrP^(Sc). These differences could either be dueto fundamental conformational differences or to the lack of GPI anchor,N-linked glycans, brain-derived ligands, or impurities in the rPrP-res.Furthermore, it is not known whether rHaPrP-res^((Sc)) is infectious, socaution should be used in interpreting conformational analyses ofrHaPrP-res^((Sc)). Nonetheless, the data indicate that rHaPrP-res^((Sc))is more closely related to bona fide HaPrP^(Sc) than isrHaPrP-res^((spon)).

Example 6 Competition Between rHaPrP-Res^((Sc)) and rHaPrP-Res^((spon))

This Example describes the competition between rHaPrP-res^((Sc)) andrHaPrP-res^((spon)) seen when reactions are seeded with bothrHaPrP-res^((Sc)) and rHaPrP-res^((spon)).

The effects of dual seeding of rPrP-PMCA reactions with bothrHaPrP-res^((Sc)) and rHaPrP-res^((spon)) were tested using differentseed ratios (FIG. 3). When the amounts of each seed were equivalent, amixture of the expected rHaPrP-res^((Sc)) and rHaPrP-res^((spon))reaction products was observed. However, when one seed concentration waskept constant, addition of the other seed reduced the formation ofproducts expected from the first type of seed. Excesses of 10- to100-fold of one seed type nearly eliminated the seeding activity of theother. This competition and/or interference between the two types ofseed makes it unlikely that, once either rHaPrP-res^((Sc)) orrHaPrP-res^((spon)) fibrils are prevalent in a reaction, the other couldovertake the reaction. This effect is probably due to competition forthe rPrP-sen substrate between mutually exclusive types of fibrils.

Example 7 Seeding with Cerebral Spinal Fluid (CSF)

This example demonstrates that CSF samples can be used to discriminateuninfected and scrapie-affected hamsters by rPrP-PMCA.

Because CSF is more accessible than brain tissue, rPrP-PMCA seedingactivity was compared in CSF samples collected from six hamsters showingclinical signs of scrapie and three uninfected control animals (allmale). After one 24-hour round, no rHaPrP-res was observed in thecontrol reactions. However, all of the scrapie CSF reactions producedthe typical rHaPrP-res^((Sc)) banding pattern with variable intensities(FIG. 4A). After second reactions seeded with 10% of the volume of thefirst round reactions, the control reactions each showed typicalrHaPrP-res^((spon)) patterns, while the scrapie-seeded reactionsproduced strong rHaPrP-res^((Sc)) patterns of relatively uniformintensity (FIG. 4B). Analysis of CSF samples from 11 additionaluninfected control hamsters (2 females and 9 males) in a 2-roundrPrP-PMCA gave either no rHaPrP-res or the rHaPrP-res^((spon)) pattern.Thus, CSF samples can be used to discriminate uninfected andscrapie-affected hamsters by rPrP-PMCA.

Example 8 QUIC

This Example demonstrates that rPrP-PMCA can be carried out in the formof an alternative assay referred to herein as QUIC (quaking-inducedconversion). In a QUIC assay, aggregates are disrupted with periodicshaking of the reaction mix, rather than (or in addition to) sonication.

Some laboratories have found the classical PMCA reaction to bechallenging to duplicate consistently, apparently due primarily todifficulties in preparing the required brain homogenate substratepreparations and delivering consistent sonication energy to multiplereactions. To circumvent the aforementioned problems with sonication,the QUIC assay was developed as a simplified and more easily replicablemethod for sensitive PrP^(Sc) and/or prion detection. Like rPrP-PMCA,QUIC uses rPrP-sen as a substrate, but substitutes periodic shaking forsonications. Even with this modification, QUIC still can beapproximately 10 times faster than the current PMCA method that usedbrain homogenate as a source of PrP-sen. The QUIC method is able todetect about 1 lethal intracerebral scrapie dose within about 8 hours,and subinfections doses with longer protocols. Under cell-freeconditions with intermittent shaking, sub-fentogram amounts of PrPSc inbrain homogenate and 2 μl cerebral spinal fluid (CSF) fromscrapie-affected hamsters seeded the conversion of recombinant prionprotein into easily detectable quantities of specific protease-resistantisoforms.

A solution of 0.2 mg/ml full-length bacterially expressed hamsterrPrP-sen (residues 23-231) was seeded with 10 ng of purified hamsterPrP^(Sc) (263K strain) and the reaction incubated for 25 hours with orwithout periodic shaking (FIG. 10A). Treatment of the reaction productswith proteinase K (PK) and immunoblotting using an antiserum (R20)raised against a C-terminal PrP epitope revealed PrP^(Sc)-seededPK-resistant conversion products (rPrP-res^((Sc))). Consistent withprevious observations with sonicated (rPrP-PMCA) reactions (describedherein), QUIC reactions produced prominent rPrP-res^((Sc)) bands of 17,13, 12 and 10 kDa. Without shaking, the same rPrP-res^((Sc)) bands wereproduced, but were much less intense.

Dilutions of scrapie brain homogenate were then seeded in normal brainhomogenate and the rPrPsen concentration and reaction volume were varied(FIG. 10B). In 20-hour reactions, 100 μl reactions with 0.2 mg/mlrPrP-sen produced the most intense rPrP-res^((Sc)) bands using seeddilutions containing as little as 10 fg PrP^(Sc). Reactions seeded withonly normal brain homogenate produced either no PK-resistant products ora spontaneously arising product(s), rPrP-res^((spon)), that gives a setof 10-13 kD PK-resistant bands. The latter were similar to thoseobserved previously in unseeded rPrP-PMCA assays as described herein.With 48-hour incubations at 0.2 mg/ml rPrP-sen, still smaller amounts ofscrapie brain homogenate seeded detectable rPrP-res^((Sc)) in both 50-and 100-μl reactions, with the latter being more sensitive (FIG. 11A).Similar to previous findings with rPrP-PMCA reactions, when the blot wasprobed with an antibody to an epitope within PrP residues 96-106 (D13),the 17-kDa band was stained preferentially. This indicated that thesmaller 10-13 kDa bands that stained with the C-terminal antibody R20were C-terminal fragments that lacked the D13 epitope. With 65- and95-hour incubations of 100 μl reactions, seed dilutions containing aslittle as 100 ag PrP^(Sc) produced strong rPrP-res^((Sc)) signals (FIG.11).

In order to further improve sensitivity, two serial rounds of QUICreactions were performed in which products of a first 48-hour round werediluted into fresh rPrP-sen for a second-round reaction (FIG. 12). Inthe first round, seeds nominally containing as little as 25-50 ag ofPrP^(Sc) were frequently positive. After second reactions seeded with10% of the volume of the first round reactions, more consistentdetection of sub-femptogram amounts of PrP^(Sc) was observed with one ofthe 10-ag seeded samples being positive for rPrP-res^((Sc)).

Because cerebral spinal fluid (CSF) is a more accessible biopsy specimenthan brain, rPrP-PMCA seeding activity was compared in CSF samplescollected from both hamsters showing clinical signs of scrapie anduninfected control animals. After one 48-hour round, no rHaPrP-res wasobserved in the control reactions. However, all of the scrapie CSFreactions produced the typical rHaPrP-res^((Sc)) banding pattern withvariable intensities (FIG. 13). After the second serial reaction rounds,the control reactions still lacked rPrP-res, while the reactions seededwith scrapie CSF produced strong rHaPrP-res^((Sc)) patterns ofrelatively uniform intensity. Thus, QUIC reactions seeded with CSFsamples can discriminate between uninfected and scrapie-affectedhamsters.

Thus, QUIC provides a simple and easily duplicated alternative tosonication for supporting an ultra-sensitive assay for prions. Withsonication of reaction tubes in cuphorn probes, the delivery ofvibrational energy to samples can vary substantially and unpredictablywith tube position, tube construction, probe age, bath volume, and theredistribution of samples within the tubes by sonication-inducedatomization and condensation. In contrast, when a group of sample tubesare shaken in a rack, each tube is subjected to the same motion, makingit easier to treat all reactions equivalently. The sonicated rPrP-PMCAreactions is somewhat faster and more sensitive than the shaken QUICreactions when both are performed at 37° C., but elevating thetemperature of the QUIC reactions improves the speed of the reaction andcan shorten the overall assay length.

The observation that the QUIC assay can discriminate between CSF samplestaken from control and scrapie-affected hamsters indicates that adiagnostic test for prion infections based on CSF samples, as opposed tobrain tissue, is feasible.

Testing of QUIC reaction conditions revealed that periodic shakingenhanced PrP^(Sc) seeded conversion of hamster rPrP-sen (residues23-231) into PK-resistant conversion products [rPrP-res(Sc), where (Sc)refers to seeding by PrP^(Sc)] (FIG. 10) which, consistent with ourprevious observations with sonicated (rPrP-PMCA) reactions 7, producedprominent rPrP-res(Sc) bands of 17, 13, 12 and 11 kDa. Periodic shakingcan therefore substitute for sonication in promoting rPrP-res(Sc)formation. The rPrP-res(Sc) generation was further improved by varyingrPrP-sen concentration, reaction volume (FIG. 10), reaction time (FIG.11), number of serial reactions (FIG. 12), temperature (FIG. 18), andshaking cycle (FIG. 19). Furthermore, addition of the detergentN-lauroyl sarcosine to the PK-digestion buffer improved the ratio of the17-kDa rPrP-res(Sc) band to the smaller bands (FIG. 20). In QUICreactions performed at 45° C., rPrP-res(Sc) formed in triplicate 1-round46-h QUIC reactions seeded with ≧100-ag of PrPSc (FIG. 14). In contrast,21 negative control reactions seeded with comparable dilutions of normalbrain homogenate or buffer alone produced no rPrP-res (FIG. 14). Resultssimilar to those in FIG. 1 were obtained in an identical repeatexperiment done in triplicate. When products of PrPSc-seeded reactionswere diluted 1000-fold into fresh rPrP-sen to seed the subsequentreaction rounds, strong propagation of rPrP-res^((Sc)) through at least4 serial reactions was observed. Under some conditions, such as withmultiple serial 48-h reaction rounds at 45° C., reactions seeded withonly normal brain homogenate occasionally generated a spontaneousproduct, rPrP-res^((spon)), indicated by a set of ≦13 kDa PK-resistantbands. The latter were similar to those observed previously in unseededrPrP-PMCA assays and were clearly distinct from the overall rPrP-res(Sc)banding profile. Hence longer amplification assays, although they candetect very small amounts of target in the sample, form more of theunwanted rPrP-res^((spon)) product that competes with the desiredamplification reaction and that product could be confused withrPrP-res^((Sc)) under some conditions.

Consistent with previous findings with rPrP PMCA reactions, we foundthat when blots of PrPSc-seeded reaction products were probed with anantibody to PrP residues 95-103 (D13)8, the 17-kDa rPrP-res(Sc) band wasstained preferentially (FIG. 10). This result indicated that the smaller11-13 kDa bands that reacted with the C-terminal antibody R20 wereC-terminal fragments lacking the N-terminal portion of PrP containingthe D13 epitope. Elevation of QUIC reaction temperatures acceleratedrPrP-res^((Sc)) formation (FIG. 18). At 55° C., rPrP-res^((Sc)) wasdetected in 8-hour reactions seeded with as little as 10 fg PrPSc (˜oneintracerebral infectious dose) (FIG. 18), while 1 fg could be detectedin triplicate 18-hours reactions (FIG. 21). At 65° C., 100 fg PrP^(Sc)seed could be detected in only 4-hours (FIG. 18). However, at 65° C.,there was also more rapid formation of rPrP-res^((spon)) in reactionsseeded with normal brain homogenate, which was apparent in all threereactions at 18 hours. Overall, there is a tradeoff between sensitivityand speed in QUIC assays and at any given temperature, the longer thetotal reaction times the greater the likelihood of spontaneous (unseededrPrP-res formation. However, spontaneous rPrP-res has usually producedpatterns of PK-resistant bands that are distinct from rPrP-res^((Sc)).Interestingly, the patterns can be altered when reaction conditions werepushed to both higher temperatures and relatively long reaction times.The QUIC reaction conditions can be altered to reduce the production ofspontaneous rPrP-res that appears similar to rPrP-res^((Sc)) accordingto the rPrP-sen sequence used in the QUIC reaction.

Cerebral spinal fluid (CSF) is a more accessible biopsy specimen thanbrain, hence QUIC seeding activity was evaluated in CSF samplescollected from hamsters showing clinical signs of scrapie or uninfectedcontrol animals. After one 48-h round (at 37° C.), no rHaPrP-res wasseen in the control reactions. However, all of the scrapie CSF reactionsproduced the distinctive rHaPrP res(Sc) banding pattern albeit withvariable intensities (FIG. 17). After a second serial QUIC reaction, thecontrol reactions still lacked rPrP-res, while the reactions seeded withscrapie CSF produced strong rHaPrP-res(Sc) patterns of similarintensity. Similar 2-round QUIC reactions showed that CSF samples from10 additional uninfected control hamsters produced no rHaPrP-res bandswhile two of the original scrapie-positive CSF samples again producedstrong rHaPrP-res(Sc) patterns (data not shown). Thus, QUIC reactionsseeded with CSF samples can discriminate between uninfected andscrapie-affected hamsters.

A QUIC assay provides a simple alternative to sonication for supportingan ultrasensitive prion assay. The delivery of vibrational energy tosamples does not vary substantially with tube position, tubeconstruction, probe age, bath volume, and the redistribution of samplesas often occurs within the tubes with sonication-induced atomization andcondensation. The 45° C. single-round QUIC reaction is virtually assensitive as 2-round sonicated rPrP-PMCA reactions of similar overallduration. The QUIC reaction conditions are also less permissive ofspontaneous unseeded rPrP-res^((spon)) formation. Significantly,elevated reaction temperatures can greatly accelerate QUIC reactions,allowing detection of a lethal dose of 263K scrapie (i.c.) in <1 day(See FIGS. 15 and 18). The relative speed, simplicity and ease ofduplication of the QUIC reaction conditions offers major practicaladvantages.

It is also possible to vary the shaking cycle to obtain surprisinglysuperior results in the QUIC assay. For example, the ratio of time spentshaking to time at rest can be varied to improve the outcome of theassay. In some examples, the ratio of time shaking:time at rest can varyfrom 1:15 to 1:1, such as 1:11 to 1:1. In particular examples, it hasbeen found that substantially equal periods of shaking and rest provideparticularly good results. For example, a shaking cycle of 60 seconds onand 60 seconds off works better than the 10 seconds on, 110 seconds offcycle for the hamster scrapie QUIC assay using rPrP-sen 23-231. Thetotal length of a cycle (time spent shaking plus time spent not shaking)may be less than about an hour, or even less than 5 minutes, for exampleless then 3 minutes, such as 2 minutes (120 seconds) or less. Inparticular examples, the total cycle is more than 60 seconds, such as60-180 seconds, or 60-120 seconds. The shaking cycle can be optimizedwith regard to the rPrP-sen sequence used in the QUIC reaction.

Example 9 Exemplary Protocol for rPrP-PCMA

This Example provides an exemplary step-by-step protocol for rPrP-PMCAusing hamster 263K scrapie seed and hamster rPrP-sen substrate. Althoughspecific exemplary protocols are provided, one will appreciate thatother similar protocols can be used.

I. Sample and Substrate Preparation

A. Preparation of Normal or 263K Scrapie Brain Homogenates (NBH andScBH, Respectively):

Reaction tubes were 0.2 ml thin wall PCR tube strips (Nalge NuncInternational 248161). Sample and substrate preparation was carried outas follows:

-   -   1) Perfuse normal or scrapie-affected Syrian golden hamsters        with ice cold 1×PBS-EDTA:

NaCl 8 g KCl 0.2 g Na₂PO₄ 1.44 g KH₂PO₄ 0.24 g +5 mM EDTA pH to 7.4 withHCl QS to 1 L

-   -   2) Extract hamster brain with clean tools and flash freeze with        liquid nitrogen    -   3) Store perfused brains at −80° C.    -   4) Dounce homogenize perfused brains, on ice, in conversion        buffer (10% weight to volume):

1X PBS-EDTA from step #1 19.3 ml  (but 1 mM EDTA) 5M NaCl 0.6 ml TritonX-100 0.1 ml Complete Protease Inhibitor Cocktail, 1 tablet/20 mls EDTAfree (Roche 11836170001)

-   -   5) Spin NBH at 2000 g for 2 minutes to partially clarify;        collect supernatant    -   6) Prepare 1 ml 10% NBH aliquots and flash freeze in liquid        nitrogen    -   7) Store aliquots at −80° C.

B. Preparation of Hamster rPrP-Sen:

Materials:

Approximately a 2 g cell pellet of rHaPrP 23-231 (yield from ¼ of 1 LLB-Miller growth medium)

BugBuster™ and lysonase bioprocessing reagent (EMD Biosciences)

8M Guanidine in water

Ni-NTA Superflow resin (Qiagen)

Denaturing Buffer: 100 mM sodium phosphate, 10 mM Tris, 6M Guanidine, pH8.0

Refolding Buffer: 100 mM sodium phosphate, 10 mM Tris, pH 8.0

Elution Buffer: 500 mM imidazole, 10 mM Tris, 100 mM Phosphate pH5.8-6.0

Dialysis Buffer: 10 mM sodium phosphate, pH 6.5 (diluted from 1M stockat pH 5.8)

AKTA Explorer 10 liquid chromatography system

Bacterial Cell Lysis:

200 μL of lysonase bioprocessing reagent and 1 Complete proteaseinhibitor tablet (Roche) were mixed into 50 mL of BugBuster™ and stirredon ice. The frozen cell pellet was sliced with a razor blade and addedportionwise into the BugBuster™ solution. Stirring was performed at 0°C. while breaking up larger pieces with a spatula. Sonication wasperformed for 15 second intervals with a Misonix ultrasonic celldisrupter (power level 10) periodically over the course of ˜30 minutesuntil the mixture was relatively homogeneous and became milky.Centrifugation was carried out at at 10,000 g (JA 12 rotor, BeckmanCentrifuge) for 10 minutes. Pellets were washed twice with 20 mLBugBuster™ diluted 10-fold with water, dispersed with pipette-aid, andcentrifuged at 10,000 g for 10 minutes. The washing, dispersing andcentrifuging was repeated, and the inclusion body pellet was stored at−20° C.

Purification was carried out by filling a 2×2 L graduated cylinder with10 mM phosphate dialysis buffer diluted from 1 M stock at pH 5.8. Allchromatography buffers were filtered prior to use. The inclusion bodypellet was dissolved into 8 mL of 8 M guanidine and mixed by pipettingup and down with a transfer pipette. The mixture was transferred into 2mL flip cap tubes and centrifuged at 8,000 g for 10 minutes. Filter andwash fresh Ni-NTA Superflow resin (Qiagen) exhaustively with water.Store dry at 4° C. Then 18 g of Ni-NTA resin was weighed into a cleanbeaker and the resin pre-equilibrated with 30-40 mL of denaturing bufferby stiffing at room temperature. Supernatant was added from 2 mL flipcap tubes to the resin and the tube discarded with the pellet. Stirringwas carried out for an additional 30 minutes. The resin slurry waspoured into an empty XK16/20 column and a column attached withimpregnated resin to an AKTA Explorer 10 (GE/Amersham) according to themanufacturer's directions. The column outlet was detached and theflow-though collected directly in a graduated cyclinder. Theflow-through can either be discarded or saved for future use, as thereis typically excess PrP in this solution.

A linear gradient was run with 0-100% refold buffer at 0.75 mL/min over5-6 hours, followed by 100% refolding buffer for 30-60 minutes at 1mL/min. The pump were rinsed with distilled water, and then Line Aequipped with refold buffer and Line B with elution buffer. The bottomof column was reattached to the UV and conductivity detector. Elutionbuffer was run through line B and the UV autozero detector set at 280nm. The refolded peptide was eluted at 2 mL/min for 20 minutes. After abrief forerun, the major fraction was collected at UV 280 as one portionin a 250 mL graduated cylinder prefilled with 50 mL pure water. Theprotein was diluted with water to 150 mL, then sterile filtered with a150 mL filter unit. An expected concentration of ˜0.1-0.15 mg/mL isdetermined by A₂₈₀. The protein was dialyzed (Snakeskin dialysis tubingMWCO 7000) overnight in dialysis buffer, and the protein transferredinto fresh dialysis buffer for 1 hour. If there was any turbidity atthis point, immediate sterile filtration was performed. The peptide wasanalyzed for purity by SDS-PAGE, Western blot, and MALDI, and theprotein concentrated to ˜0.4 mg/ml in 10 mM sodium phosphate buffer, pH6.5, using an Amicon Ultracel −10 k filter (15 ml capacity). Aliquotswere flash frozen and stored at −80° C. Once thawed, it was kept at 4°C.

C. 4×PMCA Buffer

(Final composition: 0.2% SDS, 0.2% TritonX-100, 4×PBS)

10% SDS stock (20 μl/ml)

10% TritonX-100 stock (20 μl/ml)

10×PBS stock (400 μl/ml):

Na₂HPO₄7H₂0 26.8 g/L NaH₂PO₄H₂0 13.8 g/L NaCl 75.9 g/L pH 6.9 H₂O (560μl/ml)II. rPrP-PMCA PROTOCOL:

A. 1^(st) rPrP-PMCA Round was Carried Out According to this Protocol:

1) Sonicator setup: Misonix 3000 with microplate (cup)horn accessory

2) Circulating water bath was set up at 39.4 degrees for cup horn,resulting in a temperature of 37 degrees in the cup horn.

3) 1 ml of 1×PMCA buffer was made up from 4× stock

4) Thawed aliquots of 10% NBH & 10% ScBH

5) Centrifuged 10% NBH at 1000 rcf for 5 minutes at 4° C. to removelarge debris.

6) Made up 1 ml of 1% NBH by dilution into 1×PMCA buffer

7) Prepared 263K BH seed diluted in 1% NBH (see dilution series below)

-   -   a) dilute 10% 263K BH 1:20 into 1% NBH (5 μl stock+9 μl 1%        NBH)→500 μg/1 μl    -   b) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→50 μg/1 μl    -   c) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→5 μg/1 μl    -   d) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→500 fg/1 μl    -   e) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→50 fg/1 μl    -   f) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→5 fg/1 μl    -   g) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→500 ag/1 μl    -   h) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→50 ag/1 μl    -   i) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→5 ag/1 μl    -   j) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→0.5 ag/1 μl        -   (1 fg=2 μl of g)        -   (100 ag=2 μl of h)        -   (30 ag=6 μl of i)        -   (10 ag=2 μl of i)

8) Prepared reaction mix in reaction tubes as described above (adding inthe order specified)

1^(st) Round Reaction Mix: 31.3 μl H2O   20 μl 4X PMCA buffer 26.7 μlrPrP-sen (to give a final concentration of 0.1 mg/ml)   2 μl ScBH seeddiluted in 1% NBH   80 μl total volume

9) When adding the rPrP-sen and then the seed material to the 1×PMCAbuffer in the reaction mix, mixing was performed by pipetting up anddown gently without vortexing. The reaction tubes were capped butwithout vortexing. Place tube strips were placed in a floating 96 wellrack in the sonicator cup horn, cover cup with plastic wrap to reducesplashes and evaporation.

10) Started sonicator program (typical: 40 second intermittentsonication at power setting #10, 16 minute total sonication time, 59minute 20 second incubations between each sonication, 24 hour totalcycle time)

11) Following sonication cycle (24 hours), turned off sonicator andremoved tube strips.

12) Spun the tube strips briefly to bring solution down out of the caps.

13) Removed aliquot for 2^(nd) rPrP-PMCA round and/or prepared formethanol precipitation and immunoblot analysis (see below).

B. 2nd PMCA Round:

1) Prepare reaction mix in fresh reaction tube strips as described for1^(st) round above. The sample was gently vortexed to evenly suspendjust prior to transferring volume. Following the addition of rPrP-sen,mixing was performed by pipetting up and down.

2nd Round Reaction Mix: 30 μl H₂O 18 μl 4X PMCA buffer 24 μl rPrP-sen (avolume to give a final concentration of 0.1 mg/ml)  8 μl reactionaliquot from first round rPrP-PMCA

2) The reaction tube strips were capped, and the tube strips placed inthe floating rack in the sonicator cup horn. The cup horn was coveredwith plastic wrap to reduce splashes and evaporation. Thet sonicatorprogram was started (typical: 40 second intermittent sonication at #10,16 minute total sonication time, 59 minute 20 second incubations betweeneach sonication, 24 hours total cycle time). Following the sonicationcycle (24 hours), the sonicator was turned off and tube strips removed.The tube strips were quick spun to bring solution down out of the caps,and the samples were methanol precipitated prior to further analysis(see below).

C. PK-Digestion and SDS-PAGE Sample Preparation:

(Note: In the following example, the methanol precipitation-associatedsteps 7-11 can often be omitted, in which case the products of step 6are mixed directly with more concentrated SDS-PAGE sample buffer)

1) Prepared 0.1% SDS in 1×PBS

2) Transferred 5 μl of each sample to a clean screw cap tube. (vortexedsample to evenly suspend just prior to transferring volume)

3) Added 19 μl 0.1% SDS in PBS

4) Added 1 μl 75 μg proteinase K (PK)/ml (final concentration will be 3μg PK/ml) PK storage buffer

-   -   PK storage buffer:        -   50% glycerol        -   1 mM CaCl2        -   50 mM Tris, pH 8.5

5) Incubated at 37 degrees for 1 hour

6) Added 1 μl of 0.1M PEFABLOC® (4-(2-Aminoethyl)-benzensulfonylfluoride) (Roche), vortex and place on ice

7) Added 4 μl of thyroglobulin (5 mg/ml), vortex and keep on ice

8) Added 120 μl (4 volumes) of cold methanol, vortexed and kept on ice

9) Stored at −20 degrees for >1 hour

10) Spun at 20800 rcf in the Eppendorf 5417R centrifuge at 4 degrees for30 minutes

11) Aspirated off supes and leave caps off to air dry samples

12) Added 15 μl 1×SDS-PAGE sample buffer containing 4M Urea to each tube

13) Vortexed samples in SDS-PAGE sample buffer for 1 minute

14) Boiled tubes for 10 minutes

15) Loaded sample onto 10% NUPAGE® (polyacrylamide) gel & run

D. Immunoblotting:

Wet transfer was performed using Towbin transfer buffer, IMMOBILON®-PBlotting sandwiches (transfer membrane, Millipore IPSN07852) and BIORADMINI TRANS-BLOT® for 1 hour at 0.3 amps constant. The primary antibodiesused were R20 (J. Virol. 65, 6597-6603 (1991)) at 1:30,000 or D13(Nature 412, 739-743 (2001)) at 1:10,000. The secondary antibodies wereanti-rabbit or anti-human AP conjugated, as appropriate Immunostainingwas visualized by ATTOPHOS® AP Fluorescent Substrate System (Promega)(2′-[2-benzothiazoyl]-6′-hydroxybenzothiazole phosphate [BBTP])according to the manufacturer's recommendations.

III. QUIC Protocol

A. 1^(st) QUIC Round:

1 ml of 1×PMCA buffer was made up from 4× stock, and aliquots of 10% NBH& 10% ScBH were thawed. Centrifuged 10% NBH at 2000 rcf for 2 minutes at4° C. to remove large debris. Made up 1 ml of 1% NBH by dilution into1×PMCA buffer and prepared 263K BH seed diluted in 1% NBH (see dilutionseries below).

263K BH Seed Dilution Series:

a) dilute 10% 263K BH 1:20 into 1% NBH (5 μl stock+95 μl 1% NBH)→500pg/1 μl

b) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→50 pg/1 μl

c) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→5 pg/1 μl

d) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→500 fg/1 μl

e) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→50 fg/1 μl

f) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→5 fg/1 μl

g) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→500 ag/1 μl

h) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→50 ag/1 μl

i) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→5 ag/1 μl

j) further dilute 1:10 (5 μl previous+45 μl 1% NBH)→0.5 ag/1 μl

-   -   (1 fg=2 μl of g)    -   (100 ag=2 μl of h)    -   (30 ag=6 μl of i)    -   (10 ag=2 μl of i)

Prepared reaction mix in reaction tubes as described above (add in theorder specified).

1^(st) Round Reaction Mix: 47.4 μl H2O   25 μl 4X PMCA buffer 25.6 μlrPrP-sen   2 μl ScBH seed diluted in 1% NBH  100 μl total volume

Adjusted H₂O and rPrP-sen volumes to give a final rPrP-sen concentrationof 0.1 mg/ml. When adding the rPrP-sen and then the seed material to the1×PMCA buffer in the reaction mix, mixing was performed by pipetting upand down gently without vortexing. The reaction tubes were capped butnot vortexed. The tubes were placed in Eppendorf Thermomixer R with24×0.5 ml tube block and incubated in Thermomixer R for the desired timeat 37° C., alternating between 10 seconds of shaking at 1500 rpm and noshaking for 110 seconds, unless designated otherwise. The tubes werespun to bring any solution down out of the caps. An aliquot was removedfor 2′ QUIC round and/or prepared for PK digestion, methanolprecipitation and immunoblot analysis (see below).

B. 2nd QUIC Round:

The reaction mix was prepared in fresh reaction tubes similar to Etround above. The sample tubes were gently vortexed to evenly suspend anypellet just prior to transferring volume to the 2^(nd) round reactiontube. Following the addition of rPrP-sen, mixed by pipetting up anddown.

2^(nd) Round Reaction Mix: 43.5 μl H2O 22.5 μl 4X PMCA buffer   24 μlrPrP-sen   10 μl sample volume from 1^(st) round reaction  100 μl totalvolume

The H₂O and rPrP-sen volumes were adjusted to give a final rPrP-senconcentration of 0.1 mg/ml. When adding the rPrP-sen and then the seedmaterial to the 1×PMCA buffer in the reaction mix, mixing was performedby pipetting up and down gently without vortexing. The seed was dilutedin 1% NBH as described in the 1^(st) QUIC round, and the remainder ofthe method performed as in 1^(st) QUIC round.

PK-Digestion and SDS-PAGE Sample Preparation:

(Note: In the following, the methanol precipitation-associated steps7-11 can often be omitted, in which case the products of step 6 is mixeddirectly with more concentrated SDS-PAGE sample buffer)

1) Prepared 0.1% SDS in 1×PBS

2) Transferred 10 μl of each sample to a clean screw cap tube andvortexed sample to evenly suspend any pellet just prior to transferringvolume

3) Added 38 μl 0.1% SDS in PBS

4) Added 2 μl 75 μg proteinase K (PK)/ml (final concentration will be 3μg PK/ml) PK storage buffer (50% glycerol, 1 mM CaCl₂, 50 mM Tris, pH8.5)

5) Incubated at 37 degrees for 1 hour

6) Added 1 μl of 0.1M PEFABLOC® (4-(2-Aminoethyl)-benzensulfonylfluoride) (Roche), vortexed and placed on ice

7) Added 4 μl of thyroglobulin (5 mg/ml), vortexed and kept on ice

8) Added 120 μl (4 volumes) of cold methanol, vortexed and kept on ice

9) Stored at −20 degrees for ≧1 hour

10) Spun at 20800 rcf in Eppendorf 5417R centrifuge at 4 degrees for 30minutes

11) Aspirated off supernatant and left caps off to air dry samples

12) Added 15 μl 1×SDS-PAGE sample buffer containing 4M Urea to each tube

13) Vortexed samples in SDS-PAGE sample buffer for 1 minute

14) Boiled tubes for 10 minutes

15) Loaded sample onto 10% NUPAGE® (polyacrylamide) gel & run

Immunoblotting:

Wet transferred using Towbin transfer buffer, IMMOBILON®-P Blottingsandwiches (transfer membrane, Millipore IPSN07852) and BioRad MINITRANS-BLOT® for 1 hour at 0.3 amps constant.

Primary antibodies: R20 [J. Virol. 65, 6597-6603 (1991)] at 1:30,000 orD13 [Nature 412, 739-743 (2001)] at 1:10,000.

Secondary antibodies anti-rabbit or anti-human AP conjugated, asappropriate.

Immunostaining was visualized by ATTOPHOS® AP Fluorescent SubstrateSystem (Promega) (2′-[2-benzothiazoyl]-6′-hydroxybenzothiazole phosphate[BBTP]) according to the manufacturer's recommendations.

Example 10 Amplification of PrP-Res from a Variant-CJD (vCJD) Patient

FIG. 20 shows Western blots from a QUIC reaction seeded either withdilutions of a brain homogenate (BH) from human variant CJD patient(vCJD BH) containing 100 fg, 10 fg, or 1 fg of PrP-res or, as a negativecontrol, a dilution of a non-CJD human brain homogenate (from anAlzheimer's patient; AD-BH) equivalent to the 100-fg vCJD braindilution. The recombinant PrP (rPrP-sen) substrate in these reactionswas the Syrian hamster PrP sequence (residues 23-231). A single-roundreaction was performed at 50° C. for either 8 hours (top blots) or 18hours (bottom blots). The primary antiserum used to detect therPrP-res[CJD] reaction products was R20. Six separate reactions wereperformed with each type or dilution of seed and the number ofrHaPrP-res^((vCJD))-positive reactions per 6 replicates is indicatedbelow each set of replicates on the blots.

vCJD-BH dilutions containing a nominal 100 fg of PrP-res produced clearrHaPrP-res^((vCJD)) patterns in five out of six 8-h reactions, and in6/6 18-hour cross-species QUIC reactions. Samples with 10 fg PrP-reswere positive for rHaPrP-res^((vCJD)) in 5/6 reactions of both 8 and 18h. Samples with 1 fg PrP-res were rHaPrP-res^((vCJD))-positive in ⅙ 8-hand 2/6 18-h reactions. At the same time, the AD-BH gave norHaPrP-res^((vCJD))-positive reactions with either reaction time.Although it is unknown how much PrP-res is required for an infectiousdose, it is known that a lethal intracerebral dose of hamster 263Kscrapie usually corresponds to 1-10 fg of PrP^(Sc). Thus, thiscross-species QUIC reaction can detect quantities of vCJD PrP-res (aslittle at 10 fg and even as low as 1 fg) that approximate that of aninfectious dose of scrapie by the most efficient intracerebral route.

The assay was carried out in 0.5 ml conical microcentrifuge tubes withscrew caps (Fisher 02-681-334). Brains were homogenized in conversionbuffer (10% weight to volume):

1X PBS-EDTA from step #1 19.3 ml  (but 1 mM EDTA) 5M NaCl 0.6 ml TritonX-100 0.1 ml Complete Protease Inhibitor Cocktail, 1 tablet/20 mls EDTAfree (Roche 11836170001)

Brain homogenates (BH) were spun spin at 2000 g for 2 minutes topartially clarify; supernatant was collected and 1 ml 10% AD-BH andvCJD-BH aliquots were prepared and frozen for storage at −80° C.

Hamster rPrP-sen was prepared as in Example 8, as were bacterial celllysis and purification. The same 4×PMCA buffer was used as in Example 8.The QUIC protocol was carried out by making up a working stock of 0.1%SDS in 1×PBS, thawing aliquots of 10% AD-BH & 10% vCJD-BH, making up 1ml of 1×N2 supplement (Invitrogen) by dilution into 0.1% SDS/PBS. TheAD-BH & vCJD-BH seed dilutions in 1×N2 were carried out as follows:

-   -   7.1 ul 10% AD-BH or 10% vCJD-BH+2.9 ul 1×N2→1 ug/2 ul    -   further dilute 1:10 (5 ul previous+45 ul 1×N2)→100 pg/2 ul    -   further dilute 1:10 (5 ul previous+45 ul 1×N2)→10 pg/2 ul    -   further dilute 1:10 (5 ul previous+45 ul 1×N2)→1 pg/2 ul    -   further dilute 1:10 (5 ul previous+45 ul 1×N2)→100 fg/2 ul    -   further dilute 1:10 (5 ul previous+45 ul 1×N2)→10 fg/2 ul    -   further dilute 1:10 (5 ul previous+45 ul 1×N2)→1 fg/2 ul

Recombinant PrP was filtered with a 100 kD microtube filter (PALL) byspinning at 3000×g for 12 min, and diluted 1:10 in 0.1% SDS/PBS andmeasured spectrometrically for optical density at 280 nm.

[Protein] mg/mL=(280 nm reading/PrP Extinction Coefficient(2.6))*Dilution Factor=X mg/mL

-   -   *Want 0.1 mg/mL rPrP in 100 uL reaction=10 ug/X=Y uL rPrP per        reaction    -   *Amount of water in reaction=100−Y−2−25=Z uL Water per reaction

The reaction mix was prepared in reaction tubes as described above (addin the order specified).

1^(st) Round Reaction Mix: Z ul H₂O 13 ul 25 ul 4X QUIC buffer 25 ul 2ul ScBH seed diluted in 1% NBH 2 ul Y ul rPrP-sen 60 ul 100 ul totalvolumeThe first three components were vortexed for 5s prior to adding therPrP-sen, and the rPrP-sen was added gently, as not to create bubbles.The reaction tubes were capped but not vortexed. The tubes were placedin an Eppendorf Thermomixer™ with 24×0.5 ml tube block and incubated forthe designated time (either 8 or 18 hrs) at 50° C., alternating between60 seconds of shaking at 1500 rpm and no shaking for 60 sec. TheThermomixer R is programmed to adjust to 4° C. following the 50° C.time. Spinning of the tubes was performed to recover any solution fromthe caps.

PK-digestion and SDS-PAGE sample preparation were performed by preparing1% N-lauroylsarcosine sodium salt (sarkosyl) in 1×PBS, and dilutingstock proteinase K (PK) (10 mg/ml) 100-fold into PK storage buffer(final concentration will be 100 ug PK/ml).

-   -   PK storage buffer:    -   50% glycerol    -   1 mM CaCl₂    -   50 mM Tris, pH 8.5        Further diluted 100 μg PK/ml solution above 1 to 5 in 1%        Sarkosyl/PBS (25 ul+100 ul 1% Sarkosyl/PBS), transferred 5 μl of        PK/Sarkosyl solution to a fresh set of tube, and vortexed QUIC        sample tubes evenly to suspend any pellet just prior to        transferring volume, then transferred 10 ul to individual tubes        containing PK/Sarkosyl. Incubation was performed at 37° C. for 1        hour, then 15 μl 2X SDS-PAGE sample buffer containing 4M Urea        was added to each tube. The samples were vortexed in SDS-PAGE        sample buffer for 1 minute, the tubes boiled for 10 minutes, and        the samples subjected to zip spinning and loaded onto 10%        NUPAGE® polyacrylamide gel (Invitrogen) with MES buffer        (Invitrogen).

Immunoblotting was performed by pre-incubating membranes in methanol for3 minutes to wet the PVDF membrane, pouring off the methanol and addingTowbin buffer to the VDF membrane. Dry transfer was performed usingInvitrogen iGel System and IMMOBILON-P® polyvinylidene fluoride (PVDF)membrane (Millipore IPSN07852) for 7 minutes. The membrane was blockedin 5% Milk/TBST at room temperature for 30 minutes. It was exposed toprimary antibody (R20 at 1:10,000 (2 uL/20 mL 5% Milk/TBST) for 30 minat room temperature) then washed 3× in ˜30 mL TBST (500 uL Tween 20/1 L1×TBS) for minutes per wash. The secondary antibody was Goatanti-rabbit-AP conjugate (1:10,000 in 5% milk/TBST or 2 uL/20 mL)(Jackson) for 30 minutes). Washing was performed 3× in TBST for 5minutes per wash. Then 1.5 mL ATTOPHOS® AP (alkaline phosphatase)Fluorescent Substrate System (Promega)(2′-[2-benzothiazoyl]-6′-hydroxybenzothiazole phosphate [BBTP]) wasadded to the plastic container and gel placed face down onto it for 4minutes, following which the gel was removed and left on its edge todry. The gel was visualized on a STORM™ imaging system (Amersham).

Example 11 Amplification of PrP from Sheep and Cows

Sheep with nervous disorders resembling those of a scrapie infection arepurchased or donated. In some cases, sheep are diagnosed with scrapie byhistopathological and immunohistochemical examination of the brain. Ifnecropsy is performed, it is performed within 36 hours after naturaldeath or immediately after killing the animal by intravenous injectionof sodium pentobarbital and exsanguination. The brain is removed fromeach sheep for scrapie diagnosis. Blood, serum, cerebral spinal fluidand/or brain tissue samples are obtained from each sheep.

Cows with nervous system disorders resembling those of bovine spongiformencephalitis are also tested. These animals can be “downers” or canexhibit less severe symptoms. In some cases, animals that appear healthycan be tested to determine that they are not infected.

The samples are used to seed the conversion of rPrP-sen toprotease-resistant forms in reactions performed in 0.1% sodium dodecylsulfate and 0.1% Triton X-100, in PBS at 37° C. in 0.5 ml tubes. Tubeshaking is done at 1500 rpm in an Eppendorf Thermomixer R or byvortexing. Proteinase K digestions and immunoblotting were performed asdescribed above.

For comparing PK-resistant QUIC reaction products, 24-hour unshakenreactions and reactions were shaken with or without 0.1 mm glass celldisruption beads (Scientific Industries). These reactions are seededwith 0.2 mg/ml hamster rPrP-sen, 0.2 mg/ml bovine rPrP-sen, or 0.2 mg/mlsheep rPrP-sen and a 50 μl reaction volume. The tubes are subjected tocycles of 2 minutes of shaking and 28 minutes without shaking.C-terminal antibody R20 is used for the immunoblot. The tubes aresubjected to cycles of 10 seconds of shaking and 110 seconds withoutshaking. R20 was used for the immunoblot.

If needed 65-hour and 95-hour QUIC reactions are carried out asdescribed above, and 0.2 mg/ml rPrP-sen, is used for 100-μl reactionvolumes. Cycles of 10 seconds shaking and 110 seconds without shakingcan be used.

In other examples, 48-hour reaction times are used with reduceddetergent concentrations (0.05% SDS and 0.05% Triton X-100). For thesecond round, 10% of the volume of the first round reaction products arediluted into 9 volumes of reaction buffer containing fresh rPrP-sen.PK-digestions and immunoblotting using either R20 or D13 primaryantibodies were performed as described above.

For seeding with CSF samples, aliquots (2 μl) of CSF are used to seedQUIC reactions using the conditions, and immunoblots are carried outusing the PK-digested products of the first 48-hour round. Ten percentof each first round reaction volume is used to seed a second 48-hourround of QUIC. Antibodies R20 and D13 are used for the immunoblots.

The foregoing examples provide specific examples of methods for carryingout the disclosed assay. In view of the many possible embodiments towhich the principles of the disclosed assay can be applied, it should berecognized that the illustrated embodiments should not be taken as alimitation on the scope of the disclosure. Rather, the scope of thedisclosure is defined by the following claims. We therefore claim anthat comes within the scope and spirit of these claims.

We claim:
 1. A method for detecting protease resistant prion protein(PrP-res) in a sample comprising: (a) mixing the sample with a purifiedrecombinant protease-sensitive prion protein (rPrP-sen) to make areaction mix, wherein the rPrP-sen comprises the amino acid sequence setforth as SEQ ID NO: 3, the amino acid sequence set forth as SEQ ID NO:4, or the amino acid sequence set forth as amino acids 23-231 of SEQ IDNO: 8; (b) performing an amplification reaction comprising: (i)incubating the reaction mix at about 37 to 55° C. to permitcoaggregation of the rPrP-sen with the PrP-res that may be present inthe reaction mix, and maintaining incubation conditions that promotecoaggregation of the rPrP-sen with the PrP-res and result in aconversion of the rPrP-sen to the recombinant protease resistant prionprotein initiated by the presence of prions (rPrP-res^((Sc))), which isinitiated by the presence of PrP-res in the sample, while inhibitingdevelopment of spontaneously occurring recombinant prion protein(rPrP-res^((spon))); (ii) agitating aggregates formed during step (i),in shaking cycles, wherein each shaking cycle of the shaking cyclescomprises a period of rest and a period of shaking, and wherein theperiod of rest is about 30 seconds in length and the period of shakingis about 30 seconds in length, or wherein the period of rest is about 60seconds in length and the period of shaking is about 60 seconds inlength, wherein agitating is performed for about 2 to 48 hours in theabsence of sonication; and (c) detecting rPrP-res^((Sc)) in the reactionmix, wherein detection of rPrP-res^((Sc)) in the reaction mix indicatesthat PrP-res was present in the sample.
 2. The method of claim 1,wherein detecting the rPrP-res^((Sc)) comprises detectingrPrP-res^((Sc)) aggregates in the sample.
 3. The method of claim 1,wherein the method further comprises digesting the reaction mix withproteinase K prior to detecting rPrP-res^((Sc)) in the reaction mix. 4.The method of claim 2, wherein detecting rPrP-res^((Sc)) comprisesdetecting rPrP-res^((Sc)) with an antibody that specifically binds toprion protein.
 5. The method of claim 4, wherein the antibody is apolyclonal antibody.
 6. The method of claim 4, wherein the antibody is amonoclonal antibody.
 7. The method of claim 1, wherein steps (a) and (b)are repeated for approximately 48 hours.
 8. The method of claim 1,wherein the reaction mix is incubated at 45° C. to 55° C.
 9. The methodof claim 1, wherein the reaction mix is incubated at 45° C.
 10. Themethod of claim 1, wherein the rPrP-sen consists of the amino acidsequence set forth as one of SEQ ID NO: 3, SEQ ID NO: 4, amino acids23-231 of SEQ ID NO: 8, or SEQ ID NO:
 8. 11. The method of claim 1,wherein the sample is a tissue sample from an animal.
 12. The method ofclaim 1, wherein prion can be detected in a sample containing no morethan about 1 fg PrP-res.
 13. The method of claim 1, wherein the methodis a method of diagnosing a prion disease.
 14. The method of claim 1,wherein detecting rPrP-res^((Sc)) in the reaction mix comprises the useof a fluorescence assay.
 15. A method for amplifying and detecting thehuman protease resistant prion protein (PrP-res) in a sample comprising:(a) mixing the sample with purified recombinant protease-sensitive prionprotein (rPrP-sen) comprising the amino acid sequence forth as SEQ IDNO: 8, SEQ ID NO: 3 or SEQ ID NO: 4 to make a reaction mix; (b)performing an amplification reaction comprising: (i) incubating thereaction mix at about 37° C. to 55° C. to permit formation of aggregatesof the rPrP-sen with the human PrP-res that may be present in thereaction mix, and maintaining incubation conditions that promoteaggregation of the rPrP-sen with the human PrP-res and result in aconversion of the rPrP-sen to recombinant protease resistant prionprotein initiated by the presence of prions (rPrP-res^((Sc))) whileinhibiting development of spontaneously occurring recombinant prionprotein (rPrP-res^((spon)); (ii) agitating aggregates formed during step(i), in shaking cycles, wherein each shaking cycle of the shaking cyclescomprises an equal period of rest and a period of shaking, and whereinthe shaking cycle is from about 60 to 120 seconds in length, and whereinagitating is performed in the absence of sonication; (c) detectingrPrP-res^((Sc)) in the reaction mix using fluorescence, whereindetection of rPrP-res^((Sc)) in the reaction mix indicates that PrP-reswas present in the sample.
 16. The method of claim 15, wherein thepurified recombinant protease-sensitive prion protein (rPrP-sen)comprises hamster rPrP-sen and human rPrP-sen.
 17. The method of claim15, comprising incubating the reaction mix at about 37° C., 45° C. or55° C.
 18. The method of claim 15, wherein the purified recombinantprotease-sensitive prion protein (rPrP-sen) consists of the amino acidsequence set forth as SEQ ID NO: 8, the amino acid sequence set forth asamino acids 23-231 of SEQ ID NO: 8, the amino acid sequence set forth asSEQ ID NO: 3 or the amino acid sequence set forth as SEQ ID NO:
 4. 19.The method of claim 15, wherein agitating aggregates formed during step(i), in shaking cycles, is performed for about 2 to 48 hours.
 20. Amethod for detecting Creutzfeldt-Jakob disease in a subject, comprising:(a) mixing a biological sample from the subject with purifiedrecombinant protease-sensitive prion protein (rPrP-sen) comprising theamino acid sequence forth as amino acids 23-231 of SEQ ID NO: 8 to makea reaction mix; (b) performing an amplification reaction comprising: (i)incubating the reaction mix at about 37° C. to 55° C. to permitformation of aggregates of the rPrP-sen with any human proteaseresistant prion protein (PrP-res) that may be present in the reactionmix, and maintaining incubation conditions that promote aggregation ofthe rPrP-sen with the human PrP-res and result in a conversion of therPrP-sen to recombinant protease resistant prion protein initiated bythe presence of prions (rPrP-res^((Sc)) while inhibiting development ofspontaneously occurring recombinant prion protein (rPrP-res^((spon)));(ii) agitating aggregates formed during step (i), in shaking cycles,wherein each shaking cycle of the shaking cycles comprises an equalperiod of rest and a period of shaking, and wherein the shaking cycle isabout 120 seconds in length, and wherein agitating is performed in theabsence of sonication; (c) detecting rPrP-res^((Sc)) in the reaction mixusing fluorescence, wherein detection of rPrP-res^((Sc)) in the reactionmix indicates that the subject has Cruzefeld-Jakob disease.
 21. Themethod of claim 20, wherein the sample is a blood sample or a cerebralspinal fluid sample.